Electrophoresis and Blotting
Protein Blotting Guide
BEGIN
Protein Blotting Guide
Theory and Products
Part 1 Theory and Products
5
Chapter 1 Overview of Protein Blotting
5
29
Total Protein Detection
Detection6
31
Anionic Dyes31
Fluorescent Protein Stains
31
Stain-Free Technology32
Colloidal Gold32
General Considerations and Workflow
6
Immunodetection32
Chapter 2 Methods and Instrumentation
9
Transfer6
Protein Blotting Methods
10
Electrophoretic Transfer10
Tank Blotting10
Semi-Dry Blotting11
Microfiltration (Dot Blotting)
Blotting Systems and Power Supplies
12
Tank Blotting Cells12
Mini Trans-Blot® Cell and Criterion™ Blotter
12
Trans-Blot® Cell12
Trans-Blot® Plus Cell13
Semi-Dry Blotting Cells13
Trans-Blot® SD Semi-Dry Cell
14
Trans-Blot® Turbo™ System14
Microfiltration Apparatus14
Bio-Dot® and Bio-Dot® SF Apparatus
14
Power Supplies for Electrophoretic Transfers
15
PowerPac™ HC Power Supply
15
PowerPac™ Universal Power Supply
15
Chapter 3 Membranes and Transfer Buffers 17
Membranes and Blotting Papers
18
18
18
Immun-Blot® and Immun-Blot LF PVDF for Western Blotting
18
Sequi-Blot ™ PVDF for Protein Sequencing
18
Blotting Filter Papers19
Membrane/Filter Paper Sandwiches
19
Nitrocellulose and Supported Nitrocellulose
Polyvinylidene Difluoride (PVDF) Transfer Buffers19
Towbin and Bjerrum Schafer-Nielsen Buffers
(Tris/Glycine Buffers)20
CAPS Buffer20
Discontinuous Tris-CAPS Buffer System
(for Semi-Dry Transfer)20
Dunn Carbonate Buffer21
Other Buffers21
Chapter 4 Transfer Conditions
23
General Workflow – Electrophoretic Transfer
24
Power Conditions24
Useful Equations24
Joule Heating and Other Factors Affecting Transfer
24
Relationship Between Power Settings and Transfer Times
24
High-Intensity Field Transfers24
Standard Field Transfers26
Selecting Power Supply Settings
26
Transfers Under Constant Voltage
26
Transfers Under Constant Current
26
Transfers Under Constant Power
26
General Guidelines for Transfer Buffers and
Transfer Conditions26
2
Chapter 5 Detection and Imaging
Immunodetection Workflow33
Blocking33
Antibody Incubations33
Washes33
Antibody Selection and Dilution
34
Primary Antibodies34
Species-Specific Secondary Antibodies
34
Antibody-Specific Ligands34
Detection Methods35
Colorimetric Detection36
Premixed and Individual Colorimetric Substrates
38 Immun-Blot® Assay Kits
38 Immun-Blot Amplified AP Kit
38
Opti-4CN™ and Amplified Opti-4CN Substrate
and Detection Kits38
Chemiluminescence Detection38
Immun-Star ™ Chemiluminescence Kits
40
Fluorescence Detection40
Other Detection Methods41
Bioluminescence41
Chemifluorescence42
Autoradiography 42
Immunogold Labeling42
Stripping and Reprobing42
Imaging — Analysis and Documentation
43
Luminescence Detection43
Digital Imaging for Fluorescence, Chemifluorescence,
and Colorimetric Detection
44
Autoradiography44
Analysis Software44
Part 2 Methods47
Protocols48
Transfer Buffer Formulations
58
Towbin Buffer58
Towbin Buffer with SDS
58
Bjerrum Schafer-Nielsen Buffer
58
Bjerrum Schafer-Nielsen Buffer with SDS
58
CAPS Buffer58
Dunn Carbonate Buffer58
0.7% Acetic Acid58
Detection Buffer Formulations
58
General Detection Buffers
58
Total Protein Staining Buffers and Solutions
59
Substrate Buffers and Solutions
60
Stripping Buffer60
Part 3 Troubleshooting
63
Transfer64
Electrophoretic Transfer64
Microfiltration65
Detection66
Immunodetection66
Multiscreen Apparatus68
Total Protein Detection68
Appendix70
Protein Standards for Blotting
70
71
Unstained SDS-PAGE Standards
71
Precision Plus Protein Unstained Standards
71
Prestained Standards for Western Blotting
72
Precision Plus Protein Prestained Standards
72
Kaleidoscope Standards72
Prestained SDS-PAGE Standards 72
Precision Plus Protein™ WesternC™ Standards
73
Unstained Standards for Protein Blotting
Glossary74
References and Related Reading
78
Ordering Information80
Electrophoretic Transfers48
Reagent and Materials Preparation
48
Tank Blotting Procedure49
Prepare the Gel and Membrane Sandwich
49
Assemble the Tank and Program the Power Supply
50
Semi-Dry Blotting Procedure51
Trans-Blot® Turbo™ Blotting Procedure
52
Microfiltration53
Blot Stripping and Reprobing
54
Total Protein Detection55
SYPRO Ruby Stain55
Ponceau S Stain55
Colloidal Gold Total Protein Stain
55
Immunodetection56
Notes for Multiplex Detection
56
Notes for Chemiluminescence Detection
57
Notes for Fluorescence Detection
57
Note for Protein G-HRP Detection
57
Notes for Amplified Opti-4CN™ Detection
57
Notes for Amplified AP Detection
57
3
Protein Blotting Guide
Theory and Products
PART 1
Theory and
Products
CHAPTER 1
TABLE OF CONTENTS
Overview of
Protein Blotting
Protein blotting, the transfer of
proteins to solid-phase membrane
supports, is a powerful and popular
technique for the visualization and
identification of proteins. When
bound to membranes, proteins are
readily accessible for immunological
or biochemical analyses, quantitative
staining, or demonstration of proteinprotein or protein-ligand interactions.
This chapter provides an overview of
the methods and workflow of protein
blotting, which involves two phases:
transfer and detection.
4
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Protein Blotting Guide
Theory and Products
Transfer
The first phase of protein blotting is the transfer step,
which involves moving the proteins from a solution or
gel and immobilizing them on a synthetic membrane
support (blot). Proteins can be transferred to
membranes using a number of methods but the most
common are electrophoretic transfer and microfiltration
(dot blotting). Though diffusion or capillary blotting
methods may also be used to transfer proteins from
gels, generally electrophoretic transfer is used to
transfer proteins following electrophoretic separation
by native or SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) and microfiltration is used to transfer
proteins that are in solution.
Detection
TABLE OF CONTENTS
The second phase, detection, entails probing the
membrane with either a total protein stain or primary
antibody specific to the protein of interest and
subsequent visualization of the labeled proteins.
This involves a number of steps, including the
selection of the appropriate method, reagents, and
imaging equipment.
The most commonly used protein blotting technique,
western blotting (immunoblotting), was developed as
a result of the need to probe for proteins that were
inaccessible to antibodies while in polyacrylamide
gels. Western blotting involves the transfer of proteins
that have been separated by gel electrophoresis onto
a membrane, followed by immunological detection
of these proteins. Western blotting combines the
resolution of gel electrophoresis with the specificity of
immunoassays, allowing individual proteins in mixtures
to be identified and analyzed.
Since the development of immunoblotting techniques,
other probing and detection techniques have been
developed for functional protein characterization (for
a review, see Kurien and Scofield 2003). This manual
summarizes the most commonly used techniques,
provides information about the wide selection of blotting
apparatus and detection reagents available from Bio-Rad,
and offers troubleshooting tips and technical advice.
General Considerations and Workflow
The protein blotting workflow involves selection of the
appropriate method, apparatus, membrane, buffer,
and transfer conditions. Once proteins are immobilized
on a membrane, they are available for visualization,
detection, and analysis.
Protein Blotting Workflow
Select the method
Method selection depends
largely on the starting sample
(liquid protein sample or gel)
Select the equipment
Consider the experimental approach, sample
format, and desired resolution and throughput
Prepare the reagent
Selecting the appropriate membrane and transfer buffer
is critical to successful protein transfer. Consider the
size and charge of the proteins, the transfer method, and
the binding properties of the membrane
Perform the transfer
Set up the transfer apparatus. For electrophoretic
transfer, select the transfer conditions; use the highest
electric field strength (V/cm) possible within the heat
dissipation capabilities of the system
Detect and image the protein
The choice of staining or detection technique
is determined by sensitivity requirements and the
imaging equipment available
6
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Protein Blotting Guide
Theory and Products
TABLE OF CONTENTS
CHAPTER 2
Methods and
Instrumentation
The initial step in any blotting
experiment is the selection of
transfer method and appropriate
transfer instrumentation. Method
selection depends largely on the
starting sample (liquid protein
sample or gel); the instrumentation
depends on the sample format and
desired resolution and throughput.
This chapter describes a number of
the most common techniques and
systems used today.
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Protein Blotting Guide
Theory and Products
Protein Blotting Methods
The two most common methods for protein
transfer are (Fig. 2.1):
n Electrophoretic transfer — for proteins already
separated in gels (for example, following
polyacrylamide gel electrophoresis, or PAGE),
electrophoretic transfer preserves the highresolution separation of proteins by PAGE
n
Microfiltration — for proteins in solution,
microfiltration is fast and useful for determining
working conditions for a new blotting assay or any
other situation where the resolving power of gel
electrophoresis is not needed
Electrophoretic Transfer
In electrophoretic transfer, an electric field is used
to elute proteins from gels and transfer them to
membranes. Electrophoretic transfer is the most
widely used blotting method because of its speed
and precision in replicating the pattern of separated
proteins from a gel to a membrane.
TABLE OF CONTENTS
In an electrophoretic transfer, the membrane and
protein-containing gel are placed together, with filter
paper between two electrodes (Figure 2.2). Proteins
migrate to the membrane following a current (I) that
is generated by applying a voltage (V) across the
electrodes* following Ohm’s law:
V=IxR
where R is the resistance generated by the materials
placed between the electrodes (that is, the transfer
buffer, gel, membrane, and filter papers).
The electric field strength (E, measured in V/cm) that
is generated between the electrodes is the driving
force for transfer. Both the applied voltage and the
distance between the electrodes then play a major
role in governing the rate of elution of the proteins
from the gel. A number of other factors, including the
size, shape, and charge of the protein* and the pH,
viscosity, and ionic strength of the transfer buffer, as
well as gel composition also influence the elution of
particular proteins from gels.
There are practical limits on field strength, however,
due to the production of heat during transfer. The heat
generated (Joule heating) is proportional to the power
consumed by the electrical elements (P), which is
equal to the product of the current (I) and voltage (V):
P = I x V = I2 x R
Joule heating increases temperature and decreases
resistance of the transfer buffer. Such changes in
resistance may lead to inconsistent field strength and
* Proteins denatured with sodium dedecyl sulfate (SDS) carry a net
negative charge and migrate toward the anode.
10
transfer or may cause the transfer buffer to lose its
buffering capacity. In addition, excessive heat may
cause the gel to deteriorate and stick to the membrane.
The major limitation of any electrophoretic transfer
method is the ability of the chamber to dissipate heat.
+
–
There are two main types of electrophoretic blotting
apparatus and transfer procedures (Table 2.1):
+–
+––
n Tank transfer systems — gels and membranes are
submerged under transfer buffer in tanks; these
systems are useful for most routine protein work,
for efficient and quantitative protein transfers, and
for transfers of proteins of all sizes. Tank transfer
systems offer the most flexibility in choosing voltage
settings, blotting times, and cooling options
n
Semi-dry systems — gels and membranes are
sandwiched between buffer-wetted filter papers
that are in direct contact with flat-plate electrodes;
these systems are typically easier to set up than
tank systems and are useful when high-throughput
is necessary and extended transfer times are not
required or when discontinuous buffer systems are
used. Active cooling options are limited with semidry blotting
Tank Blotting
In tank blotting systems, the gel and membrane
sandwich is entirely submerged under transfer buffer
within a buffer tank. A nonconducting cassette holds
the membrane in close contact with the gel and the
cassette assembly is placed in the tank between
the electrodes, transverse to the electrical field
and submerged under conducting transfer buffer
(Burnette 1981, Gershoni et al. 1985, Towbin et al.
1979). Although the large volumes of buffer in the
tank dissipate the heat generated during transfer and
provide the conducting capacity for extended transfer
conditions, additional cooling mechanisms are offered
by the various tank blotter systems.
Semi-Dry Blotting
In a semi-dry transfer, the gel and membrane are
sandwiched between two stacks of filter paper that
are in direct contact with plate electrodes (Bjerrum
and Schafer-Nielsen 1986, Kyhse-Andersen 1984,
Tovey and Baldo 1987). The term “semi-dry” refers to
the limited amount of buffer, which is confined to the
two stacks of filter paper.
In semi-dry systems, the distance between the
electrodes is limited only by the thickness of the gel
and membrane sandwich. As a result, high electric
field strengths and high-intensity blotting conditions
are achieved. Under semi-dry conditions, some small
proteins may be driven through the membrane in
response to the high field strengths. Moreover, because
low buffer capacity limits run times, some large proteins
++
––
MICROFILTRATION (DOT BLOTTING)
ELECTROPHORETIC TRANSFER
TANK TRANSFER
–+
++ -
SEMI-DRY TRANSFER
Fig. 2.1. Protein transfer methods.
Table 2.1. Comparison of electrophoretic protein transfer systems.
Tank Blotting
Semi-Dry Blotting
Traditional
15–60 min
Rapid
Transfer time
30 min–overnight
3–10 min
Handling convenience
Manual assembly of transfer
Manual assembly of transfer
Prepackaged, presaturated componentscomponents components
Transfer parameters
Widest range of power settings Power and transfer time limited
and transfer times
due to lack of cooling options
Preinstalled, customizable programs
for transfers of most proteins,
user-programmable settings for
traditional semi-dry techniques
Molecular weight range Broad range
Best for 30–120 kD Broad range
Temperature control
Cooling with ice pack or refrigerated water recirculator None
None
Buffer requirement
1–12 L, system-dependent 250 ml per blot
No additional buffer required
Cathode (–)
Filter paper
Gel
Membrane
Filter paper
Anode (+)
Fig. 2.2. Gel and membrane setup for electrophoretic transfer.
may be poorly transferred. Use of a discontinuous buffer
system (see Chapter 3) may enhance semi-dry transfer
of high molecular weight proteins (>80 kD). As semidry transfers require considerably less buffer and are
easier to set up than the tank method, laboratories
performing large numbers of blots often favor them.
Novel buffer and material formulations have been
developed that can be used with higher electric field
strengths than those used in typical semi-dry blotting.
These conditions yield complete and extremely rapid
transfer, with some systems completing transfer in
3–10 min. Such rapid blotting systems do not
incorporate external cooling mechanisms, so the high
power dissipation may generate more heat than other
techniques. Rapid blotting systems are intended for
extremely rapid transfers where heat-induced protein
denaturation will not affect downstream applications.
Microfiltration (Dot Blotting)
Simple bulk transfer of proteins that are in solution
may be achieved by manual application (dotting) to
a membrane from a pipet or syringe, or by vacuumassisted microfiltration. Manual dot-blotting with a
pipet or syringe is generally used for small sample
volumes. Microfiltration devices, on the other hand,
enable application of larger volumes, multiple assays
with different probes, and quick, reproducible
screening of a large number of samples. Microfiltration
facilitates the determination of working conditions for
a new blotting assay and is a convenient method in
any other situation where the resolving power of gel
electrophoresis is not needed.
Links
Mini-PROTEAN ® Tetra Cell
Mini-PROTEAN ® TGX™ Gels
Gel Doc™ EZ Imager
11
Protein Blotting Guide
Theory and Products
Blotting Systems and Power Supplies
Table 2.2. Specifications for Bio-Rad’s tank blotting cells.
Buffer tank and lid
Once the transfer method has been selected, choose the
appropriate transfer cell or apparatus for that application.
Tank Blotting Cells
The tank transfer systems offered by Bio-Rad are
described below, and their specifications are summarized
in Table 2.2. Selection of the appropriate system is largely
dictated by the gel format used for separation and the
desired throughput.
Blue cooling unit
Buffer tank and lid — the buffer tank and lid
combine to fully enclose the inner chamber during
electrophoresis. On the inside, the tank has slots
for placement of the electrode cards, gel holder
cassettes, and cooling element. Ports on the lid
allow connection points for the electrodes and are
energized using an external power supply
TABLE OF CONTENTS
n Gel holder cassette — the gel and membrane
sandwich is held together between two foam pads
and filter paper sheets, and placed into the tank
within a gel holder cassette. Cassettes are made of
nonconducting material and are designed to permit
unimpeded flow of current and buffer through the
gel and membrane sandwich
n
Electrodes — tank transfer systems use either
plate or wire electrode cards. Plate electrodes offer
greater field strength than wire electrodes but wire
electrodes may be more economical and generate
less heat
n Cooling mechanism — cooling systems consist of
an in ice block, a sealed ice unit, or a cooling coil
that is coupled to an external cooling mechanism.
These cooling systems prevent temperature
fluctuations and overheating during high-intensity,
extended, or native protein transfers
Mini Trans-Blot ® Cell and Criterion™ Blotter
The Mini Trans-Blot cell and the Criterion blotter
accommodate mini- and midi-format gels. The Mini
Trans-Blot cell (Figure 2.3) can transfer up to two mini
gels (10 x 7.5 cm) in an hour and is available either as
a complete apparatus or as a module that uses the
buffer tank and lid of the Mini-PROTEAN® Tetra cell for
operation. The Criterion blotter (Figure 2.4) can transfer
up to two Criterion gels (15 x 9.4 cm) or four mini gels
in 30–60 min. A self-contained Bio-Ice™ cooling unit
absorbs the heat generated during transfer in the
Mini Trans-Blot cell, and the Criterion blotter uses a
sealed ice block or optional cooling coil to regulate
temperature during transfer.
Mini Trans-Blot®Criterion™ Blotter
Blotting area
10 x 7.5 cm
Gel holder cassette
and foam pads
Electrode assembly
Fig. 2.3. Mini Trans-Blot cell.
Buffer tank and lid
Optional cooling coil
Plate electrodes
Wire electrodes
Assembly tray with
roller, foam pads,
blotting filter paper, and
gel holder cassettes
Fig. 2.4. Criterion blotter.
Trans-Blot ® Cell
The Trans-Blot cell (Figure 2.5) offers a choice of plate or
wire electrodes and variable placement of the electrodes
for both standard and high-intensity blotting options.
The Trans-Blot cell accommodates three gel holder
cassettes, each with a 16 x 20 cm blotting area. Use
this system for transfer of large-format gels or of multiple
mini- or midi format gels. Standard field transfers are
performed with the electrodes placed 8 cm apart; with
this arrangement, all three of the gel holder cassettes
can be used simultaneously. High-intensity transfers are
performed with the electrodes placed 4 cm apart, with
a single gel holder cassette between them. Temperature
regulation can be achieved using the super cooling
coil (included) and a refrigerated water recirculator
(purchased separately).
28 x 26.5 cm
2
3
3
1.2 L
1.3 L
3–4 L
10–12 L
Electrode distance
4 cm
4.3 cm
2 positions: 4 and 8 cm
3 positions: 4, 7, and 10 cm
Platinum-coated titanium anode with stainless-steel cathode plates or platinum wire
Platinum-coated titanium
anode with stainless-steel
cathode plates or
platinum wire
Platinum-coated titanium
anode and stainless-steel
cathode plates
Electrode materials
Platinum wire
Transfer time
Wire electrodes
Standard: 16 hr
Standard: 60 min to
High-intensity: 1 hr
overnight
Standard: 5 hr
Overnight: 16 hr
High-intensity:
30 min–4 hr
Plate electrodes
Standard: 30 min
to overnight
Standard: 1–5 hr
Overnight: 16 hr
High-intensity: 30 min–1 hr
Sealed ice block or Super cooling coil
optional Criterion blotter
cooling unit
Overall dimensions 12 x 16 x 18 cm
21.8 x 11.8 x 15 cm
18 x 9.5 x 24 cm
(W x L x H)
Standard: 16 hr
High-intensity: 15 min–1 hr
Super cooling coil
30 x 17.3 x 39.4 cm
Table 2.3. Specifications for Bio-Rad’s semi-dry blotting cells.
Trans-Blot ® Plus Cell
With a 28 x 26.5 cm blotting area, the Trans-Blot Plus cell
(Figure 2.6) has the capacity to transfer three large-format
gels or multiple smaller format gels simultaneously in
as little as 15–30 min. Plate electrodes provide a strong
and uniform electrical field and are movable — up to
three gel cassettes can be placed in the tank with the
minimum electrode distance between them, increasing
the field strength and efficiency of transfer. A cooling
coil coupled to a refrigerated water recirculator provides
temperature regulation.
Trans-Blot® SD
Trans-Blot® Turbo™
Blotting area 24 x 16 cm
15 x 11 cm
Gel capacity
2 PROTEAN II gel sandwiches, stacked
and separated by dialysis membrane; 4 Mini-PROTEAN gels side by side; 3 Criterion
gels side by side
2 midi gels
(13.5 x 8.5 cm),
4 mini gels
(7 x 8.5 cm)
or similar
Transfer time
~30 min
3–10 min
Semi-Dry Blotting Cells
Electrode
dimensions
25 x 18 cm
16 x 12 cm
Electrode
distance
Determined by thickness of the gel and membrane
sandwich and filter
paper stack
~8 mm depending
on gel thickness
Electrode
materials
Platinum-coated titanium anode and stainless-steel cathode
Platinum-coated
titanium anode and
stainless-steel cathode
Semi-dry transfers allow fast, efficient, economical
blotting without a buffer tank or gel cassettes. Semidry systems do not offer external cooling. See Table
2.3 for detailed specifications.
Buffer tank and lid
Super cooling coil
Buffer tank and lid
Gel holder cassette
and foam pads
Super cooling coil
16 x 20 cm
3 PROTEAN® II xi, 6 Criterion, Three 26.5 x 28 cm gels or
or 12 Mini-PROTEAN gels
12 Criterion gels
Buffer requirement
Buffer requirement 200 ml
N/A
Links
CoolingN/A
N/A
Overall dimensions 37 x 24 x 11 cm
(W x L x H)
26 x 21 x 20 cm
Mini-PROTEAN Tetra Cell
Mini-PROTEAN ® TGX ™ Gels
Mini Trans-Blot Cell
Criterion Blotter
Trans-Blot Cell
Trans-Blot Plus Cell
Plate electrodes
Plate electrodes
Wire electrodes
Fig. 2.5. Trans-Blot cell.
12
15 x 9.4 cm
Cooling
Blue cooling unit
Sealed ice block
Trans-Blot®Trans-Blot® Plus
Gel capacity
2 Mini-PROTEAN® gels 4 Mini-PROTEAN or 2 Criterion™ gels
Number of gel holders2
Tank transfer systems contain the following elements:
n
Fig. 2.6. Trans-Blot Plus cell.
Gel holder cassette
and fiber pads
Trans-Blot SD
Semi-Dry System
Trans-Blot Turbo System
13
Protein Blotting Guide
Theory and Products
microfiltration manifold. Each apparatus is available as
an independent unit containing both the microfiltration
manifold and the sample template, and also as a
modular template without the manifold base.
Trans-Blot ® SD Semi-Dry Cell
The Trans-Blot SD semi-dry cell (Figure 2.7) performs
electrophoretic transfers in less than 30 min. Plate
electrodes and a single-step locking system make
assembly easy and ensure uniform contact across the
entire electrode surface.
Bio-Dot microfiltration unit
The Bio-Dot® and Bio-Dot SF units can be easily
sterilized by autoclaving or by washing in alcohol or
sodium hydroxide. The units feature a unique, patented
sealing gasket that eliminates lateral leakage and
possible cross-contamination among wells. Both
sample templates are spaced similarly to microplates,
so samples can be applied with a standard or a
multichannel pipet. Specifications for the Bio-Dot units
are listed in Table 2.4.
Lid
Cathode plate
Bio-Dot SF microfiltration unit
Anode plate
Fig. 2.9. Microfiltration apparatus.
Fig. 2.7. Trans-Blot SD cell.
Trans-Blot ® Turbo™ System
TABLE OF CONTENTS
The Trans-Blot Turbo system (Figure 2.8) performs
semi-dry transfers in as little as 3 min. The system
uses prepackaged transfer packs containing a
prewet membrane (nitrocellulose or polyvinylidene
difluoride [PVDF]) and filter paper stacks soaked with a
proprietary buffer. The base unit contains an integrated
power supply that drives two independent transfer
cassettes, allowing transfer of a total of four miniformat or two midi-format gels.
Samples are loaded into the wells of the templates
and proteins are trapped on the membrane by filtration
using either vacuum or gravity flow. Once samples are
loaded, incubations, wash steps, and detection may
all be performed without removing the membrane from
the unit.
The 96-well Bio-Dot apparatus performs traditional
dot-blot comparisons and the 48-well Bio-Dot
SF apparatus focuses the applied samples into
thin lines instead of circles (Figure 2.10). The slot
format makes it easier to use a densitometer for
quantitation. The Bio-Dot and Bio-Dot SF sample
templates are interchangeable; each uses the same
1 2 3 4 5 6 7 8 9 10
A
Table 2.4. Bio-Dot apparatus specifications.
Bio-Dot
Bio-Dot SF
Sample format
96-well, 8 x 12 format
48-slot, 6 x 8 format
Well size
3 mm diameter
7 x 0.75 mm
Sample volume
50–600 μl
50–500 μl
Membrane size 9 x 12 cm
(W x L)
9 x 12 cm
AutoclavabilityYes
Yes
Power Supplies for Electrophoretic Transfers
Electrophoretic transfer cells require high currents that
not all power supplies are equipped to deliver. Table
2.5 compares the two Bio-Rad power supplies
that accommodate the needs of electrophoretic
transfer systems.
Table 2.5. PowerPac™ HC and PowerPac Universal power supply
specifications.
PowerPac HC
PowerPac Universal
Voltage
5–250 V
5–500 V
Current
0.01–3.0 A
0.01–2.5 A
Power
1–300 W
1–500 W
PowerPac HC Power Supply
B
C
Fig. 2.8. Trans-Blot Turbo system.
D
Microfiltration Apparatus
Microfiltration units use easy, reproducible methods for
binding proteins in solution onto membranes.
®
Bio-Dot and Bio-Dot SF Apparatus
The Bio-Dot and the Bio-Dot SF (slot-format)
microfiltration units (Figure 2.9) provide reproducible
binding of proteins in solution onto membranes.
14
1
2
3
4
5
6
Fig. 2.10. Multiple sample comparisons are simplified with the
Bio-Dot and Bio-Dot SF microfiltration units. A and B, antigen (human
transferrin) applied to nitrocellulose in each row of the Bio-Dot apparatus.
1, 100 ng; 2, 50 ng; 3, 25 ng; 4, 10 ng; 5, 5 ng; 6, 2.5 ng; 7, 1 ng; 8,
0.5 ng; 9, 0.25 ng; 10, 1% BSA in TBS. C and D, antigen applied to each
row of the Bio-Dot SF apparatus. 1, 100 ng; 2, 50 ng; 3, 10 ng; 4, 5 ng;
5, 1 ng; 6, 0.1 ng. The membranes were incubated with rabbit anti-human
transferrin. In A and C, Bio-Rad’s goat anti-rabbit gold conjugate and
gold enhancement kit were used to visualize the antigen. In B and D,
Bio-Rad’s goat anti-rabbit AP conjugate and the color development
reagents BCIP and NBT were used to visualize the antigen.
Fig. 2.11. PowerPac HC power supply.
The PowerPac HC (high current) power supply (Figure
2.11), is capable of driving all transfer cells to their
maximum performance. The PowerPac HC power
supply offers high power output and the flexibility of
choosing transfer under constant voltage, constant
current, or constant power settings. The PowerPac
HC power supply also offers highly regulated voltage
settings, fine adjustment of current limits, and a
convenient pause function. Safety features include
overload/short circuit detection, automatic crossover,
arc and ground leak detection, programmable
multistep methods, and a programmable timer.
Fig. 2.12 PowerPac Universal power supply.
PowerPac™ Universal Power Supply
The PowerPac Universal power supply (Figure 2.12)
is designed to drive all of the most common
electrophoretic applications, with the exception of
high-voltage applications such as isoelectric focusing
and DNA sequencing. Like the PowerPac HC power
supply, the PowerPac Universal power supply provides
the choice of transfer under constant voltage, constant
current, or constant power settings with all of the
other features listed above. In addition, the PowerPac
Universal stores up to nine methods, each with up to
nine steps, and is equipped to enable wireless transfer
of run data and protocols for instrument validation
for regulatory purposes (for example, installation
qualification and operational qualification, or IQ/OQ).
Links
Trans-Blot Turbo System
Trans-Blot Turbo
Transfer Packs
Bio-Dot Microfiltration
Apparatus
Trans-Blot SD System
PowerPac HC Power Supply
PowerPac Universal Power
Supply
15
Protein Blotting Guide
Theory and Products
TABLE OF CONTENTS
CHAPTER 3
Membranes and
Transfer Buffers
Selecting the appropriate
membrane and buffer is critical to
successful protein transfer. The
size and charge of the proteins, the
transfer method, and the binding
properties of the membrane all
must be considered. This chapter
provides technical information and
advice for selecting among the
various conditions that are available
for protein transfer.
16
17
Protein Blotting Guide
Theory and Products
Membranes and Blotting Papers
A variety of membrane types is available, each offering
key attributes to suit particular experimental conditions.
Evaluate the physical properties and performance
characteristics of a membrane when selecting a
membrane for your application (Table 3.1). Membranes
are commonly available in two pore sizes:
n
n
0.45 μm pore size membranes are recommended for
most analytical blotting experiments
0.2 μm pore size membranes are most suitable for
transfer of low molecular weight (<15,000 kD) proteins
that might move through larger membrane pores
Nitrocellulose and Supported Nitrocellulose
TABLE OF CONTENTS
Nitrocellulose was one of the first membranes used
for western blotting and is still a popular membrane
for this procedure. Protein binding to nitrocellulose
is instantaneous, nearly irreversible, and quantitative
to 80–100 μg/cm2. Nitrocellulose is easily wetted
in water or transfer buffer and is compatible
with a wide range of protein detection systems.
Unsupported nitrocellulose is innately fragile and is not
recommended for stripping and reprobing.
Supported nitrocellulose is an inert support structure
with nitrocellulose applied to it. The support structure
gives the membrane increased strength and resilience.
Supported nitrocellulose can withstand reprobing and
autoclaving (121°C) and retains the ease of wetting and
protein binding features of nitrocellulose.
Polyvinylidene Difluoride (PVDF)
PVDF membrane is an ideal support for N-terminal
sequencing, amino acid analysis, and immunoassay
of blotted proteins. PVDF retains proteins during
exposure to acidic or basic conditions and in the
presence of organic solvents. Greater protein retention
during sequencing manipulations enhances the
likelihood of obtaining information from rare, lowabundance proteins by increased initial coupling and
higher repetitive yields. In addition, PVDF membrane
exhibits better binding efficiency of electroblotted
material in the presence of SDS in the transfer buffer.
PVDF membrane must be wetted in 100% methanol
prior to use but once wet may be used with a transfer
buffer that contains no methanol. Bio-Rad offers
PVDF membrane specifically designed for protein
sequencing and for immunodetection. Both are
available in precut sheets, rolls, and sandwich formats.
Table 3.2. Guide to precut membranes and filter paper.
Blotting Cells
Precut Membranes Precut Blot Filter
Papers
Mini Trans-Blot® cell
7 x 8.5 cm
7.5 x 10 cm
Criterion™
8.5 x 13.5 cm
9.5 x 15.2 cm
Immun-Blot PVDF membrane retains target protein but
resists nonspecific protein binding that can obscure
high-sensitivity chemiluminescence and colorimetric
detection. Immun-Blot PVDF has a strong binding
capacity of 150–160 μg/cm2 (roughly twice that
of nitrocellulose), will not crack or tear in common
handling, and can withstand repeated stripping
and reprobing.
Trans-Blot® cell
13.5 x 16.5 cm
15 x 20 cm
Trans-Blot Plus cell
26.5 x 28 cm
26.5 x 28 cm
Trans-Blot SD cell
7 x 8.5 cm
11.5 x 16 cm
15 x 15 cm
15 x 9.2 cm
20 x 20 cm
15 x 15 cm
(extra thick)
Trans-Blot® Turbo™
7 x 8.5 cm and 8.5 x 13.5 cm
Transfer packs include precut membrane
and filter paper
Bio-Dot® apparatus
9 x 12 cm
N/A
Bio-Dot SF apparatus
9 x 12 cm
11.3 x 7.7 cm
Immun-Blot LF PVDF membranes combine the
advantages of Immun-Blot PVDF membranes with low
autofluorescence across a wide range of excitation and
emission wavelengths. This low autofluorescence allows
longer exposure times without increasing background
fluorescence levels, allowing fluorescent detection of
faint signals.
Sequi-Blot™ PVDF for Protein Sequencing
Sequi-Blot PVDF membrane withstands the conditions
of N-terminal sequencing while providing the binding
capacity to sequence even low-abundance samples.
Pore Size Binding Capacity (µg/cm2) Compatible Detection Methods Notes
Nitrocellulose 0.45 µm
80–100
Colorimetric
General-purpose protein blotting membrane
0.2 µm
Chemiluminescence
Chemifluorescence
Fluorescence
Radioactive
18
Blotting filter paper, made of 100% cotton fiber,
provides a uniform flow of buffer through the gel and
contains no additives that might interfere with the
transfer process. Precut filter paper is available in a
wide range of convenient sizes to eliminate waste and
save time (Table 3.2). Extra thick absorbent filter paper
is recommended for semi-dry transfers because of its
additional fluid capacity.
Immun-Blot® and Immun-Blot LF PVDF for Western Blotting
Table 3.1. Guide to protein blotting membranes.
Membrane
Blotting Filter Papers
Supported 0.45 µm
80–100
Colorimetric
nitrocellulose 0.2 µm
Chemiluminescence
Chemifluorescence
Fluorescence
Radioactive
Pure nitrocellulose cast on an inert synthetic
support; increased strength for easier handling
and for reprobing
Immun-Blot
0.2 µm
150–160
Colorimetric
PVDF
Chemiluminescence
Radioactive
High mechanical strength and chemical
stability; recommended for western blotting
Immun-Blot LF 0.45 µm
155–300
PVDF
Colorimetric
Chemiluminescence
Chemifluorescence
Fluorescent
High mechanical strength and chemical
stability; low autofluorescence; recommended
for western blotting using fluorescent detection
0.2 µm
170–200
Sequi-Blot™
PVDF
Colorimetric
Radioactive
High mechanical strength and chemical
stability; recommended for protein sequencing
blotter
Transfer Buffers
Different gel types and blotting applications call for
different transfer buffers (Tables 3.3 and 3.4), but in
general, transfer buffer must enable both effective
elution of proteins from the gel matrix and binding of
the protein to the membrane. The choice of buffer
depends on the type of gel and membrane being
used as well as the physical characteristics of the
protein of interest.
Transfer buffers contain a conductive, strong buffering
agent (for example, Tris, CAPS, or carbonate) in order
to maintain the conductivity and pH of the system
during transfer. In addition, alcohol (for example,
methanol or ethanol) may be included in the transfer
buffer to promote binding of proteins to membranes,
and SDS may be added to promote elution of proteins
from gels.
Regardless of the transfer buffer selected, when
preparing and using transfer buffers:
n
Do not use the same batch of transfer buffer more
than once, as the buffer will likely lose its capacity to
maintain a stable pH during transfer
Do not dilute transfer buffers; this will also decrease
buffering capacity
n
Do not adjust the pH of transfer buffers when not
indicated, as this increases buffer conductivity,
which is manifested by higher initial current output
and decreased resistance
n
Membrane/Filter Paper Sandwiches
Precut and preassembled sandwiches save time
and effort during western blot preparation. In
Bio-Rad’s membrane sandwiches, a precut membrane
(nitrocellulose or PVDF) and two sheets of 100%
cotton-fiber thick filter paper are preassembled into a
blotting membrane/filter paper sandwich.
Recipes for all of the buffers described in this section
are provided in Part 2 of this guide.
A Note About SDS and Alcohol
SDS and alcohol play opposing roles in a transfer.
SDS in the gel and in the SDS-protein complexes
promotes elution of the protein from the gel but
inhibits binding of the protein to membranes. In
cases where certain proteins are difficult to elute
from the gel, SDS may be added to the transfer
buffer to improve transfer. SDS in the transfer
buffer decreases the binding efficiency of protein
to nitrocellulose membrane; PVDF membrane
can be substituted for nitrocellulose when SDS
is used in the transfer buffer. Addition of SDS
increases the relative current, power, and heating
during transfer and may affect the antigenicity of
some proteins.
Alcohol (methanol or ethanol), on the other hand,
removes the SDS from SDS-protein complexes
and improves the binding of protein to
nitrocellulose membrane but has some negative
effects on the gel itself. Alcohol may cause a
reduction in pore size, precipitation of some
proteins, and some basic proteins to become
positively charged or neutral. All of these factors
will affect blotting efficiency.
Note: Only high-quality, analytical grade
methanol should be used in transfer buffer;
impure methanol can increase transfer buffer
conductivity and result in poor transfer.
Links
Nitrocellulose Membrane,
0.45 μm
Nitrocellulose Membrane,
0.2 μm
Supported Nitrocellulose
Membrane, 0.45 μm
Supported Nitrocellulose
Membrane, 0.2 μm
Immun-Blot PVDF Membrane
Sequi-Blot PVDF Membrane
19
Protein Blotting Guide
Theory and Products
CHAPTER 3
Table 3.3. General guidelines for transfer buffer and membrane selection by gel type.
Gel Type
Transfer Buffer
Membrane
SDS-PAGE
Towbin with or without SDS, CAPS,
carbonate, Bjerrum Schafer-Nielsen
Nitrocellulose, supported
Tank blotting or semi-dry blotting
nitrocellulose, or PVDF (0.45 or 0.2 μm)
Tris-Tricine
Towbin, CAPS
Nitrocellulose, supported nitrocellulose, or PVDF (0.2 μm)
Two-dimensional
Towbin with or without SDS, CAPS,
carbonate, Bjerrum Schafer-Nielsen
Notes
Tank blotting recommended; needs
high-capacity, small pore-size
membrane; pH of buffer may be critical
Nitrocellulose, supported
Tank blotting or semi-dry blotting
nitrocellulose, or PVDF (0.45 or 0.2 μm)
Native, nondenaturing Depends on pH of gel buffer and
Nitrocellulose or PVDF (0.45 or 0.2 μm) Tank blotting recommended;
pI of protein of interest
temperature regulation may be needed
to maintain activity
Acid urea
0.7% acetic acid
Nitrocellulose (0.45 or 0.2 μm)
Tank blotting or semi-dry blotting;
use acid-gel transfer protocol
(membrane toward cathode)
Isoelectric focusing
0.7% acetic acid
Nitrocellulose, supported
Tank blotting or semi-dry blotting;
nitrocellulose, or PVDF (0.45 or 0.2 μm) use acid-gel transfer protocol
(membrane toward cathode)
Table 3.4. General guidelines for transfer buffer and membrane selection by application.
Application
Transfer Buffer
Membrane
Protein sequencing
Towbin*, CAPS
Nitrocellulose, 0.45 or 0.2 μm, or PVDF Tank blotting recommended
Notes
TABLE OF CONTENTS
High molecular weight Towbin with SDS
Nitrocellulose, 0.45 or 0.2 μm, or PVDF
proteins
Tank or rapid semi-dry blotting
recommended; needs high-capacity,
small pore-size membrane;
pH of buffer may be critical
Small proteins and
Towbin, CAPS
Nitrocellulose, 0.2 μm, or PVDF
peptides Tank or rapid semi-dry blotting
recommended; pH of buffer may be
critical
Basic proteins (pI >9) in denaturing gels
CAPS, carbonate, Bjerrum
Nitrocellulose, 0.45 or 0.2 μm, or PVDF Tank blotting, semi-dry blotting, or
Schafer-Nielsen
rapid semi-dry blotting
Basic proteins (pI >9) in native or
nondenaturing gels
0.7% acetic acid
Nitrocellulose, 0.45 or 0.2 μm, or PVDF Tank blotting recommended
Glycoproteins
Towbin with or without SDS, CAPS, carbonate, Bjerrum SchaferNielsen nondenaturing gels
Nitrocellulose, 0.45 or 0.2 μm, or PVDF Tank blotting or semi-dry blotting
Proteoglycans
Towbin, Bjerrum Schafer-Nielsen
Nitrocellulose, 0.45 or 0.2 μm, or PVDF Tank blotting or semi-dry blotting
*Towbin buffer may be used for protein sequencing but extra care must be exercised to rinse Tris and glycine from the membrane after transfer.
Towbin and Bjerrum Schafer-Nielsen Buffers
(Tris/Glycine Buffers)
The most common transfers are from SDS-PAGE
gels using the buffer systems originally described
by Towbin (1979). Standard Towbin buffer contains
25 mM Tris, 192 mM glycine, pH 8.3, 20% (v/v)
methanol and, occasionally, 0.025–0.1% (w/v) SDS.
A buffer similar in composition to the standard Towbin
buffer is the Bjerrum Schafer-Nielsen buffer
(48 mM Tris, 39 mM glycine, pH 9.2, 20% methanol),
which was developed for use in semi-dry applications.
CAPS Buffer
CAPS-based transfer buffer (10 mM CAPS, pH 11, 10%
methanol) may be preferable for transfers of high molecular
weight proteins (for example, >50 kD) and in cases where
the glycine component of Towbin buffer may interfere with
downstream protein sequencing applications.
Discontinuous Tris-CAPS Buffer System
(for Semi-Dry Transfer)
A unique feature of semi-dry blotting is the ability
to use two different buffers during transfer; this is
known as a discontinuous buffer system. In a semidry transfer, the buffer reservoirs are the filter paper
on either side of the gel, which are independent
(discontinuous). In a discontinuous system, methanol
is included in the buffer on the membrane (anode)
side of the blot assembly and SDS is used on the gel
(cathode) side, taking advantage of the positive effects
of each buffer component. A discontinuous buffer
system using a Tris-CAPS buffer can greatly increase
the efficiency of protein transfer by semi-dry blotting.
This system uses 60 mM Tris, 40 mM CAPS, pH 9.6,
plus 15% methanol in the filter paper on the anode side
and 0.1% SDS on the cathode side. Concentrated,
premixed anode and cathode buffers are available
for purchase. For more information about the use of
a discontinuous buffer system in semi-dry transfer,
request bulletin 2134.
Dunn Carbonate Buffer
In some cases, using a carbonate buffer (10 mM
NaHCO3, 3 mM Na2CO3, pH 9.9, 20% methanol) may
produce higher efficiency transfers and improve the
ability of antibodies to recognize and bind to proteins.
Carbonate buffer has also been recommended for the
transfer of basic proteins (Garfin and Bers 1989).
Other Buffers
The mobility of proteins during electrophoretic transfer
from native gels will depend on the size and pI of the
protein of interest relative to the pH of the buffer used.
n
n
If the pI of the protein is greater than the pH of the
transfer buffer, the protein carries a positive charge
and will migrate toward the negative electrode
If the pI of the protein is close to the pH of the
transfer buffer, the migration of the protein out of
the gel is decreased. Use a more basic or acidic
buffer to increase protein mobility
Proteins in native gels, as well as acidic and neutral
proteins, require buffers that do not contain methanol.
Gels for isoelectric focusing, native PAGE, and those
containing basic proteins or acid-urea may be transferred
in 0.7% acetic acid. When using acetic acid for transfer,
the proteins will be positively charged, so the membrane
should be placed on the cathode side of the gel.
Links
10x Tris/Glycine
10x Tris/CAPS
10x Tris/Glycine/SDS
10x Phosphate Buffered Saline
20x SSC
20
21
Protein Blotting Guide
Theory and Products
TABLE OF CONTENTS
CHAPTER 4
Transfer Conditions
This chapter provides an overview
of the transfer conditions required
for performing electrophoretic
protein transfer. Detailed protocols
and advice for each transfer
method are available in Part 2 of
this guide.
22
23
Protein Blotting Guide
Theory and Products
General Workflow — Electrophoretic Transfer and changes in resistance may lead to inconsistent
Overall, the procedures and principles for semi-dry
and tank transfers are the same. Gels and membranes
are prewet and equilibrated with transfer buffer, and
the gel/membrane sandwich is placed into the transfer
apparatus in the correct orientation to ensure transfer
of proteins to the membrane. For electrophoretic
transfers, the appropriate power conditions must
also be selected.
field strength and transfer, may cause the transfer
buffer to lose its buffering capacity, or may cause
the gel to melt and stick to the membrane. Under
normal running conditions, the transfer buffer absorbs
most of the heat that is generated; during extended
transfer periods or high-power conditions, active
buffer cooling is required to prevent uncontrolled
temperature increases.
Power Conditions
The following variables also change the resistance of
the transfer system and, therefore, also affect transfer
efficiency and current and voltage readings:
For best transfer results, use the highest electric
field strength (E) possible within the heat dissipation
capabilities of the system. For most proteins, the
most rapid transfer occurs under conditions where
the applied voltage (V) is maximized and the distance
between the electrodes is minimized. Though rapid
blotting experiments may seem to be the most
convenient, a number of factors must be considered
when choosing the appropriate power conditions for a
given electrophoretic transfer.
Useful Equations
TABLE OF CONTENTS
Two basic equations are important in electrophoresis.
The first is Ohm’s law, which relates the applied
voltage (V) with the current (I) and resistance (R) of
the system:
V=IxR
The applied voltage and current are determined by the
user and the power supply settings; the resistance is
inherent in the system.
The second equation, the power equation, describes
the power (P) used by a system, which is proportional
to the voltage (V), current (I), and resistance (R) of the
system.
P = I x V = I 2 x R = V2 /R
Understanding the relationships among power,
voltage, current, resistance, and heat is central to
understanding the factors that influence the efficiency
and efficacy of transfer.
Joule Heating and Other Factors Affecting Transfer
The power that is dissipated is also equivalent to the
amount of heat, known as Joule heating, generated
by the system. According to the power equation, the
amount of Joule heating that occurs depends on the
conductivity of the transfer buffer used, the magnitude
of the applied field, and the total resistance within the
transfer system. During an electrophoretic transfer,
the transfer buffer warms as a result of Joule heating.
Consequently, its resistance drops. Such heating
24
General Workflow for Electrophoresis Transfer
Prepare transfer buffer
Prepare transfer buffer sufficient
for the transfer cell and for
equilibration of gels and membranes
n Alterations to buffer composition; that is, addition of
SDS or changes in ion concentration due to addition
of acid or base to adjust the pH of a buffer
n Gel pH, ionic strength, and percentage of
acrylamide, especially if the gel has not been
properly equilibrated
n Number of gels (current increases slightly as the
number of gels increases)
Equilibrate gels and membranes
Equilibrate gels and membranes
in transfer buffer
nVolume of buffer (current increases when volume
increases)
n Transfer temperature (current increases when
temperature increases)
Relationship Between Power Settings and Transfer Times
In theory, increasing the power input and duration of
an electrophoretic transfer results in the transfer of
more protein out of a gel. In practice, however, test
runs should be used to evaluate transfer efficiency at
various field strengths (by modulating both power input
and, if applicable, interelectrode distance) and transfer
times for each set of proteins of interest. The optimum
transfer conditions depend on a number of factors,
including the size, charge, and electrophoretic mobility
of the protein, the type of transfer buffer used, and the
type of transfer system being used.
High-Intensity Field Transfers
As their name suggests, high-intensity field transfers
use high-strength electrical fields that are generated by
increased voltage and closer positioning of electrodes.
High-intensity transfers often produce satisfactory
transfer of proteins in less time than standard transfers;
however, in some cases the high field strength
causes small proteins to be transferred through the
membrane. In addition, high molecular weight proteins
and other proteins that are difficult to transfer may
not have sufficient time to be transferred completely.
Because more heat is generated in high-intensity field
transfers than in standard field transfers, a cooling
device may be needed.
Assemble the gel and membrane sandwich
Place the membrane and gel
between buffer-soaked filter papers
Set up the transfer cell
Place the gel, filter paper, and membrane sandwich
in the transfer cell. Fill the cell with transfer buffer and add the
cooling unit. Connect the cell to the power supply
and set the power supply for optimal power and time
Start the transfer
25
Protein Blotting Guide
Theory and Products
Standard Field Transfers
Transfers Under Constant Voltage
Standard field transfers require less power input
and more time to complete; they are generally run
overnight. Standard transfers often produce more
complete, quantitative transfer of proteins across a
broad molecular weight range; the slower transfer
conditions allow large proteins sufficient time to move
through the gel matrix while the lower intensity allows
smaller proteins to remain attached to the membrane
after transfer.
If the voltage is held constant throughout a transfer,
the current in most transfer systems increases as
the resistance drops due to heating (the exception
is most semi-dry systems, where current actually
drops as a result of buffer depletion). Therefore, the
overall power increases during transfer, and more
heating occurs. Despite the increased risk of heating,
a constant voltage ensures that field strength remains
constant, providing the most efficient transfer possible
for tank blotting methods. Use of the cooling elements
available with the various tank blotting systems should
prevent problems with heating.
Tank transfer systems offer the capacity for both
high-intensity and standard-field transfers. Increased
buffering capacity and additional cooling mechanisms
enable longer transfer times than are feasible with
semi-dry transfers. Some tank transfer systems offer
flexible electrode positions that, when combined with
variable voltages, provide a choice of high-intensity,
rapid transfer or longer, more quantitative transfer over
a broad range of molecular weights.
TABLE OF CONTENTS
Semi-dry transfers, on the other hand, are necessarily
rapid and of high intensity. In a semi-dry transfer
system, the distance between electrodes is
determined only by the thickness of the gel-membrane
sandwich, and buffering and cooling capacity is
limited to the buffer in the filter paper. As a result, the
field strength is maximized in semi-dry systems, and
the limited buffering and cooling capacity restricts
the transfer time. Though power conditions may be
varied with the power supply, semi-dry transfers often
operate best within a narrow range of settings.
Selecting Power Supply Settings
Power supplies that are used for electrophoresis hold
one parameter constant (either voltage, current, or
power). The PowerPac™ HC and PowerPac Universal
power supplies also have an automatic crossover
capability that allows the power supply to switch over
to a variable parameter if a set output limit is reached.
This helps prevent damage to the transfer cell. During
transfer, if the resistance in the system decreases as a
result of Joule heating, the consequences are different
and depend on which parameter is held constant.
Transfers Under Constant Current
If the current is held constant during a run, a decrease
in resistance results in a decrease in voltage and
power over time. Though heating is minimized,
proteins are transferred more slowly due to decreased
field strength.
Table 4.1. Guide to power setttings for different gel types.
SDS-PAGE Gels (Towbin Buffer)
Standard (Overnight)
High-Intensity
Trans-Blot® cell
Plate electrodes
Wire electrodes
10 V/100 mA, 16 hr
30 V/100 mA, 16 hr
50–100 V/700–1,600 mA, 30–60 min
100–200 V/300–800 mA, 30 min–4 hr
Trans-Blot Plus cell
30 V/0.5 A, 16 hr
100 V/1,500 mA, 60 min
Mini Trans-Blot® cell
30 V/90 mA, 16 hr
100 V/350 mA, 60 min
Criterion™ blotter
Plate electrodes
Wire electrodes
10 V/50–80 mA, 16 hr
10 V/30–40 mA, 16 hr
100 V/750–1,000 mA, 30 min
100 V/380–500 mA, 60 min
Trans-Blot SD cell
N/A
Mini gels: 10–15 V/5.5 mA/cm2, 10–30 min
Large gels: 15–25 V/3 mA/cm2, 30–60 min
Trans-Blot® Turbo™
N/A
Mini gels: 25 V/1,300 mA, 7 min
Midi gels: 25 V/2,500 mA, 7 min
Isoelectric Focusing Gels, Native Gels, Basic Proteins, and Acid-Urea Gels (0.7% acetic acid)
Standard (Overnight)
High-Intensity
Trans-Blot cell
Plate electrodes
Wire electrodes
15 V/200 mA, 16 hr
30 V/200 mA, 16 hr
30–60 V/600–1,000 mA, 30–60 min
100–150 V/550–850 mA, 30 min–4 hr
Trans-Blot Plus cell
10–30 V/0.15–0.55 A, 16 hr 100–125 V/1.9–2.4 A, 15–60 min
If the power is held constant during a transfer, changes
in resistance result in increases in current, but to a
lesser degree than when voltage is held constant.
Constant power is an alternative to constant current
for regulating heat production during transfer.
Mini Trans-Blot cell
30 V/10 mA, 16 hr
100 V/350 mA, 1 hr
Criterion blotter
Plate electrodes
Wire electrodes
10 V/50 mA, 16 hr
10 V/50 mA, 16 hr
100 V/980–1,200 mA, 30 min
100 V/500–800 mA, 30 min
Trans-Blot SD cell
N/A
Mini gels: 10–15 V/5.5 mA/cm2, 10–30 min
Large gels: 15–25 V/3 mA/cm2, 30–60 min
General Guidelines for Transfer Buffers and Transfer Conditions
Trans-Blot Turbo
N/A
Transfers Under Constant Power
Different transfer apparatus, when used with different
gel and buffer systems, require different power
settings. Table 4.1 provides general guidelines for
the voltage and current settings recommended for
selected gel and buffer systems. Increase transfer
times for gradient gels and decrease transfer times for
low molecular weight proteins. The values presented in
Table 4.1 are guidelines — transfer conditions should
be optimized for every transfer application. Cooling
is generally required for all high-intensity transfers
(except when using the Trans-Blot® SD cell) and is
recommended for long, unsupervised runs.
Mini gels: 25 V/1,300 mA, 7 min
Midi gels: 25 V/2,500 mA, 7 min
Links
Mini Trans-Blot Cell
Criterion Blotter
Trans-Blot Cell
Trans-Blot Plus Cell
Trans-Blot SD
Semi-Dry System
Trans-Blot Turbo
Transfer System
PowerPac HC Power Supply
PowerPac Universal
Power Supply
26
27
Protein Blotting Guide
Theory and Products
TABLE OF CONTENTS
CHAPTER 5
Detection and Imaging
Total protein detection and immunodetection can be performed using
colorimetric, chemiluminescence,
and fluorescence development and
imaging techniques.
28
29
Protein Blotting Guide
Theory and Products
Anionic dyes
100–1,000 ng
Fluorescent stains
2–8 ng
Once proteins have been transferred to a membrane,
they can be visualized using a variety of specialized
detection reagents (Figure 5.1). Total protein stains
allow visualization of all the proteins on the blot while
immunological detection (immunodetection) methods
employ antibody or ligand conjugates for visualization
of specific proteins of interest. This chapter reviews the
various total protein stains and immunological detection methods available.
Total Protein Detection
Stain-free technology
Colloidal gold
Total protein staining provides an image of the
complete protein pattern on the blot (Figure 5.2). This
information helps determine transfer efficiency and
the molecular weight, relative quantity, and other
properties of the transferred proteins.
2–28 ng
0.1 pg–1 ng
Table 5.1. Comparison of total protein staining methods.
Method
Sensitivity Advantages
DisadvantagesImaging
Anionic dyes
100–1,000 ng Inexpensive, rapid Low sensitivity, Photography with
(Ponceau S, Coomassie
shrink membrane epi-illumination or
Brilliant Blue R-250, amido
reflectance densitometry
black, Fast Green FCF)
Fluorescence
2–8 ng
Sensitive, mass Fluorescence
spectrometry-
detection system
compatible
required
Stain-free
2–28 ng
Fluorescence
visualization with UV,
LED epi-illumination, or
laser scanning
Rapid – no Special gels
Gel Doc™ EZ system
additional staining and imaging
or destaining equipment
requiredrequired
Colloidal gold
100 pg–1 ng Very sensitive, Expensive
(enhanced)
rapid; optional enhancement
increases
sensitivity
Photography with
epi-illumination or
reflectance densitometry
A
Anionic Dyes
The first techniques developed for total protein staining
of blotted membranes used the same anionic dyes
commonly used for staining proteins in polyacrylamide
gels. These dyes include amido black (Towbin et al.
1979), Coomassie (Brilliant) Blue R-250 (Burnette 1981),
Ponceau S, and Fast Green FCF (Reinhart and Malamud
1982). Of these:
Total protein detection
TABLE OF CONTENTS
Immunological detection
B
Chemiluminescence
detection
HRP
fg–pg
AP
10 pg
HRP
5–500 pg
AP
10–100 pg
Amido black destains rapidly in acetic acid/
isopropanol solution and produces very little
background staining. Amido black may interfere with
downstream immunodetection.
Coomassie (Brilliant) Blue may show high
background staining, even after long destaining
procedures, and is not compatible with subsequent
immunodetection.
Colorimetric detection
Fluorescence
Other
1 pg–1 ng
Bioluminescence
Fig. 5.2. Total protein and immunological detection. A, blot stained with
SYPRO Ruby blot stain showing the total protein pattern of an E. coli lysate
containing an overexpressed GST fusion protein on the blot. B, same blot
probed for the GST-fusion protein in the lysate and detected using the
Immun-Star™ WesternC™ chemiluminescence kit.
Table 5.1 compares the advantages and
disadvantages of several total protein staining
techniques. When performing total protein blot
staining, note that:
n
Chemifluorescence
Autoradiography
n
Immunogold labeling
Fig. 5.1. Protein detection systems.
30
Protein standards are useful for monitoring transfer
efficiency and serve as molecular weight markers
for calibration of blot patterns. For information
about protein standards that are useful in blotting
applications, refer to the Appendix in this guide
Polyacrylamide gels shrink during staining,
so comparison of an immunologically probed
membrane to a stained gel is not practical. To
determine the exact location of a specific antigen
in relation to other proteins, compare two blotted
membranes, one that has been probed with an
antibody and the other stained for total protein
Ponceau S and Fast Green are compatible with
downstream immunodetection methods, and Fast
Green can be easily removed after visualization to
allow subsequent immunological probing.
These dyes are easy to prepare and they stain proteins
quickly, but they are relatively insensitive when compared
to other stains (Table 5.1). The stains that require alcoholcontaining solutions for solubility (for example, amido
black, Coomassie Brilliant Blue, and Fast Green FCF)
can shrink nitrocellulose membranes, making direct
comparison of an immunologically detected antigen to
the total protein on the stained membrane difficult.
Coomassie Blue R-250 stain is available from Bio-Rad.
Fluorescent Protein Stains
Fluorescent stains such as SYPRO Ruby and Deep Purple
provide highly sensitive detection of proteins on blots as
well as in gels. SYPRO Ruby blot stain allows detection as
low as 2 ng. After staining, target proteins can be detected
by colorimetric or chemiluminescence immunodetection
methods, or analyzed by microsequencing or mass
spectrometry with no interference from the protein stain.
Links
Coomassie Brilliant Blue R-250
SYPRO Ruby Protein Gel Stain
31
Protein Blotting Guide
Theory and Products
Stain-Free Technology
A haloalkane compound in Mini-PROTEAN® TGX
Stain-Free™ and Criterion™ TGX Stain-Free™ gels
covalently binds to protein tryptophan residues
when activated with UV light. This allows protein
detection (with a Gel Doc™ EZ imager) in a gel both
before and after transfer, as well as total protein
detection on a blot when using wet PVDF membranes.
Stain-free technology is compatible with downstream
immunodetection, though some antibodies may
show a slightly lower affinity for the haloakanemodified proteins.
Colloidal Gold
TABLE OF CONTENTS
Colloidal gold is an alternative to anionic dyes that
provides detection sensitivities rivaling those of
immunological detection methods (Moeremans et al.
1987, Rohringer and Holden 1985). When a solution
of colloidal gold particles is incubated with proteins
bound to a nitrocellulose or PVDF membrane, the
gold binds to the proteins through electrostatic
adsorption. The resulting gold-protein complex
produces a transient, reddish-pink color due to the
optical properties of colloidal gold. This gold-protein
interaction is the basis for total protein staining with
colloidal gold as well as for specific, immunogold
detection (see Immunogold Labeling on page 42).
Immunodetection
Immunodetection Workflow
Immunodetection (immunological detection) is used
to identify specific proteins blotted to membranes. The
steps used for immunological detection vary little; the
steps are summarized in Figure 5.3.
After the proteins have been transferred to the
membrane, the membrane is blocked, incubated
with a primary antibody, washed, incubated with a
secondary antibody, and washed again (Figure 5.3).
The primary antibody is specific for the protein
of interest and the secondary antibody enables
its detection. The secondary antibody can be
radiolabeled, labeled with a fluorescent compound or
gold particles, or conjugated to an enzyme such as
alkaline phosphatase (AP) or horseradish peroxidase
(HRP) for subsequent detection.
For many years, radiolabeled secondary antibodies
were the method of choice for detection, but newer
methods have evolved that are less hazardous and
easier to use than radioactivity, yet maintain the same
degree of sensitivity. Available detection methods
now also include colorimetric, chemiluminescence,
fluorescence, bioluminescence, chemifluorescence,
and immunogold detection.
Bio-Rad’s stain-free technology allows direct
visualization, analysis, and documentation of
protein samples in PAGE gels and on blots,
without staining or destaining. It also provides
equal or better sensitivity compared to
Coomassie staining and eliminates organic
waste disposal concerns.
The stain-free system comprises the Gel Doc EZ
imager with stain-free tray, Image Lab™ software,
and unique precast gels (Criterion™ and
Mini-PROTEAN® formats) that include unique
trihalo compounds that allow rapid fluorescent
detection of proteins — without staining.
The trihalo compounds in the gels react with
tryptophan residues in a UV-induced reaction
to produce fluorescence, which can be easily
detected by the imager either within gels or on
low-fluorescence PVDF membranes. Activation
of the trihalo compounds in the gels adds 58 Da
moieties to available tryptophan residues and is
required for protein visualization. Proteins that
do not contain tryptophan residues cannot be
32
Transfer proteins to a membrane
▼
detected using this system. The sensitivity of the
system is comparable to staining with Coomassie
(Brilliant) Blue for proteins with a tryptophan content
>1.5%; sensitivity superior to Coomassie staining is
possible for proteins with a tryptophan content >3%.
Benefits of stain-free technology include:
n
n
n
n
n
Elimination of staining and destaining steps for
faster results
Automated gel and blot imaging and analysis
No background variability (as is often seen with
standard Coomassie staining)
Reduced organic waste through elimination of
the need for acetic acid and methanol in staining
and destaining
Visualization of transferred (blotted) proteins on
low-fluorescence PVDF membranes
Gel
Blot
A typical immunodetection experimental system
utilizes two rounds of antibody incubation:
n
Block unbound membrane sites
▼
Incubate with primary antibody
n
▼
Wash away excess primary antibody
▼
The primary antibody, which is directed against
the target antigen; the antigen may be a ligand on
a protein, the protein itself, a specific epitope on a
protein, or a carbohydrate group
The secondary antibody, which recognizes
and binds to the primary antibody; it is usually
conjugated to an enzyme such as AP or HRP,
and an enzyme-substrate reaction is part of the
detection process (Figure 5.4)
S
Incubate with conjugated secondary antibody or ligand
4 Substrate reagent is then
added to the blot
▼
Wash away excess secondary antibody
▼
P
3
Develop signal based on the detection method
▼
A blotting grade antibodyenzyme conjugate is added
to bind to the primary
antibody
5 The enzyme catalyzes
the substrate (S) to form a
detectable product (P) at
the site of the antigenantibody complex
Document and analyze results
Fig. 5.3. Immunodetection workflow.
Blocking
Bio-Rad’s colloidal gold total protein stain provides
sensitivity to 100 pg of protein.
Stain-Free Technology
Antibody Incubations
Following transfer, unoccupied binding sites on the
membranes must be blocked to prevent nonspecific
binding of probes; failure to completely block these
sites can lead to high background. A variety of
blocking reagents is available, including gelatin, nonfat
milk, and bovine serum albumin (BSA), which are
compared in Table 5.2. Optimize the detection system
for minimal background with no loss of signal by
testing several blocking agents. The type of membrane
also affects the selection of blocker. Formulations for
different blocking solutions are available in Part 2 of
this guide.
Table 5.2. Comparison of blocking reagents.
Blocking
Reagent
Membrane Recommended
CompatibilityConcentration Notes
Gelatin
Nitrocellulose 1–3%
Requires heat to
solubilize
Nonfat dry
Nitrocellulose, 0.5–5%
milk, BLOTTO, PVDF
blotting-grade blocker
PVDF requires
higher
concentrations
of nonfat milk
than nitrocellulose
Bovine serum Nitrocellulose, 1–5%
albumin (BSA) PVDF
PVDF requires
higher
concentrations
of BSA than
nitrocellulose
Tween 20
Nitrocellulose 0.05–0.3%
May strip some
proteins from
the blot
2
Primary antibody to
a specific antigen is
incubated with the membrane
1
Blocking reagent blocks
unoccupied sites on the
membrane
Fig. 5.4. Specific enzymatic detection of membrane-bound antigens.
Antibody incubations are generally carried out in
antibody buffer containing Tris-buffered saline with
Tween (TTBS) and a blocking reagent. Various
formulations of antibody buffer are provided in Part 2
of this guide.
Washes
Washing the blot between the two antibody
incubations and prior to detection removes excess
antibody and prevents nonspecific binding. Though
washing solutions and times may vary (depending
on the antibodies and detection systems used),
washes generally involve Tris-buffered saline (TBS) or
phosphate-buffered saline (PBS). A detergent such
as Tween 20 may be added to decrease background,
but detergents may inhibit certain detection reactions
(see the instruction manuals for details). Wash buffer
formulations are described in Part 2 of this guide.
Links:
Criterion TGX Stain-Free Gels
Gel Doc EZ Imager
Colloidal Gold Total
Protein Stain
Mini-PROTEAN II
Multiscreen Apparatus
PharosFX and PharosFX
Plus Systems
Image Lab Software
33
Protein Blotting Guide
Theory and Products
Antibody Selection and Dilution
An antibody is an immunoglobulin protein such as
IgG that is synthesized by an animal in response
to exposure to a foreign substance, or antigen.
Antibodies have specific affinity for the antigens that
elicited their synthesis. Structurally, most IgG class
antibodies contain four polypeptide chains (two
identical heavy chains of ~55 kD and two identical
light chains of ~25 kD) oriented in a “Y” shape
(Figure 5.5). These are held together by disulfide
bridges and noncovalent interactions. These proteins
contain an Fab region with specific affinity for the
antigens that elicited their synthesis. In addition, a
constant region (Fc ) on the antibody provides binding
sites for proteins needed during an immune response.
en
tig site
An ing
d
bin
Variable
bin Anti
din gen
g
sit
e
Constant
Fab
TABLE OF CONTENTS
Light chain
Fc
Heavy chain
Determine the appropriate concentration or dilution
(titer) of the primary antibody empirically for each
new lot of primary antibody. The Mini-PROTEAN®
II multiscreen apparatus and mini incubation trays
(described in the sidebar on page 35) are useful tools
for determining antibody titer.
Species-Specific Secondary Antibodies
Primary Antibodies
The primary antibody recognizes and binds to the
target antigen on the membrane. For incubations with
primary antibody, the entire blot must be covered with
antibody-containing solution. The optimum antibody
concentration is the greatest dilution of antibody that
still yields a strong positive signal without background
or nonspecific reactions. Instructions for antibodies
obtained from a manufacturer typically suggest a
starting dilution range. For custom antibodies or for
those where a dilution range is not suggested, good
starting points are:
n 1:100–1:1,000 dilution of the primary antibody in
buffer when serum or tissue culture supernatants
are the source of primary antibody
n 1:500–1:10,000 dilution of chromatographically
purified monospecific antibodies
n 1:1,000–1:100,000 dilution may be used when
ascites fluid is the source of antibody
34
Table 5.3. Immunoglobulin-binding specificities of protein A
and protein G.
Immunoglobulin
Protein A
Human IgG1
••
••
—
••
•
••
••
••
•
—
••
••
•
••
—
••
—
•
•
••
•
•
—
••
Human IgG2
Human IgG3
Human IgG4
Mouse IgG1
Secondary antibodies are specific for the isotype
(class) and the species of the primary antibody (for
instance, a goat anti-rabbit secondary antibody is an
antibody generated in goat for detection of a primary
antibody generated in a rabbit). Secondary antibodies
bind to multiple sites on primary antibodies to increase
detection sensitivity. For immunodetection, use only
blotting-grade species-specific secondary antibodies.
The major limitation of protein A and protein G
conjugates is their lower sensitivity. Because only
one ligand molecule binds to each antibody, the
enhancement of a multiple-binding detection system,
such as a species-specific polyclonal antibody, is lost.
Generally, the species-specific antibody is 10–50 times
more sensitive than the ligand reagent when the same
detection system is used.
Mouse IgG2
Secondary antibodies can be labeled and detected in
a variety of ways. The antibody can be radiolabeled or
linked to a fluorescent compound or to gold particles,
but most commonly the antibody is conjugated to an
enzyme, such as alkaline phosphatase or horseradish
peroxidase. If the secondary antibody is biotinylated,
biotin-avidin-AP or -HRP complexes can be formed.
Addition of a suitable enzyme substrate results in
production of a colored precipitate or fluorescent or
chemiluminescent product through dephosphorylation
(by AP) or oxidation (by HRP).
Detection Methods
Bovine IgG2
Since the purity of the reagents is critical to the
success of the experiment, the following steps are
critical if the antibodies used are not blotting-grade:
Fig. 5.5. Antibody structure. The components of a typical IgG molecule
are highlighted and include the Fab fragment containing the variable region
responsible for antigen binding and the Fc, constant region, necessary for
binding other proteins involved in the immune response.
from many different species or for those using one of
the less common primary antibody systems in their
experiments; that is, rat, goat, or guinea pig. In addition,
these reagents bind only to antibody molecules; this
can reduce the background from nonspecific binding of
antibodies to membrane-bound proteins when a lowtiter, poorly purified second antibody is used.
n Purify all sera by affinity chromatography to obtain
only those antibodies directed against the particular
IgG; otherwise, background staining and false
positive reactions due to nonspecific antibody
binding may occur
n Cross-adsorb the purified antibody solution against
an unrelated species; for example, human IgG for
anti-rabbit and anti-mouse antibodies, and bovine
IgG for anti-human reagents, to remove antibodies
that are not specific for the species of interest
Blotting-grade antibodies are directed to both heavy
and light chains of the IgG molecules, so the
reagents can be used to identify other classes and
subtypes of immunoglobulins.
Antibody-Specific Ligands
Protein A and protein G are bacterial cell surface
proteins that bind to the Fc regions of immunoglobulin
molecules (Akerstrom et al. 1985, Boyle and Reis
1987, Goding 1978, Langone 1982). The advantage
of using protein A or protein G is their ability to bind to
antibodies of many different species (Table 5.3). This is
often desirable for laboratories using antibody probes
Blotted proteins are generally detected using secondary
antibodies that are labeled with radioisotopes or
colloidal gold, or that are conjugated to fluorescent
molecules (fluorophores) or an enzyme such as AP or
HRP. Early blotting systems used 125I-labeled reagents
similar to those used in radioimmunoassay. These
systems provide sensitive results but the special
handling and disposal problems of 125I reagents have
discouraged continued use of this technique. Instead,
a number of enzyme systems and detection reagents
have evolved (Figure 5.6).
Mouse IgG3
Mouse IgG4
Rat IgG1
Rat IgG2a
Rat IgG2b
Rat IgG2c
Pig IgG
Rabbit IgG
Bovine IgG1
Sheep IgG1
Sheep IgG2
Goat IgG1
Goat IgG2
Horse IgG(ab)
Horse IgG(c)
Horse IgG(t)
Dog IgG
•• = Strong binding
• = Weak binding
Protein G
••
••
••
••
•
••
••
••
•
••
•
••
••
••
••
••
••
••
••
••
••
••
•
•
— = No binding
Screening Antibodies
In some experiments, protein blots must be screened for a number of
different antigens or under a number of different conditions.
Mini Incubation Trays
Mini incubation trays allow safe, simple, and economical screening of
different antigens on protein blot strips. Each tray has eight 10.5 cm x
5 mm channels to accommodate strips cut from a particular protein blot.
Because the trays are disposable, the potential contamination associated
with washing reusable trays is eliminated. Ribs in the tray lids combine
with the overall design of the sample channels to ensure that no crosscontamination occurs.
Mini-PROTEAN®
Mini incubation tray.
II Multiscreen Apparatus
When proteins are resolved by SDS-PAGE and blotted onto a membrane
for analysis, the Mini-PROTEAN II multiscreen apparatus simplifies the
screening process. Instead of being cut into individual strips for incubation,
the entire blot is simply clamped into the multiscreen unit for assay. Two
separate, detachable sample templates allow up to 40 different antibody
or serum samples to be screened. The unique molded gasket ensures a
leakproof seal, preventing cross-contamination among samples.
Multiscreen apparatus.
Links:
Mini-PROTEAN II Multiscreen
Apparatus
Mini Incubation Trays
35
Protein Blotting Guide
Theory and Products
A. Colorimetric
Colorimetric Detection
B. Chemiluminescence
Substrate
Substrate
Light
Product
Product
C. Fluorescence
Light
Fig.5.6. Mechanism of detection
chemistries. In each method of
western blot detection, a detectable
signal is generated following binding
of an antibody specific for the
protein of interest. In colorimetric
detection (A), the signal is a colored
precipitate. In chemiluminescence
(B) the reaction itself emits light.
In fluorescence detection (C),
the antibody is labeled with a
fluorophore.
TABLE OF CONTENTS
The most commonly used detection methods
use secondary antibodies conjugated to alkaline
phosphatase or horseradish peroxidase. With these
methods, when the enzyme substrate is added,
either a colored precipitate is deposited on the blot
(colorimetric detection) or a chemiluminescent or
fluorescent product is formed and the light signal
is captured on film or with a digital imaging system
(Figure 5.6). Secondary antibodies conjugated to
fluorophores are gaining popularity and can be directly
visualized on the blot and captured with a compatible
imager, without the need for additional liquid
substrate (see the sidebar Fluorescence Detection
on page 40).
Enzymes such as AP and HRP convert several
substrates to a colored precipitate (Table 5.4). As the
precipitate accumulates on the blot, a visible colored
signal develops that is visible on the blot (Figure 5.6A).
The enzyme reaction can be monitored and stopped
when the desired signal over background is produced.
Colorimetric detection is easier to use than any
film-based detection method, which must be developed
by trial and error and uses costly materials such as X-ray
film and darkroom chemicals. Colorimetric detection is
considered a medium-sensitivity method, compared to
radioactive or chemiluminescence detection.
Colorimetric HRP systems — the first enzyme
conjugates used for immunological detection
of blotted proteins. The advantage of HRP
systems was that both the enzyme conjugate and
colorimetric detection substrates were economical.
The most common color substrates for HRP are
4-chloro-1-naphthol (4CN) (Hawkes et al. 1982) and
3,3’-diaminobenzidine (DAB) (Tsang et al. 1985)
(Figure 5.7). HRP colorimetric detection systems
are not as sensitive as AP colorimetric detection
systems. Fading of blots upon exposure to light,
inhibition of HRP activity by azide, and nonspecific
color precipitation are additional limitations of HRP
colorimetric detection systems
HRP + H2O2
36
NH2
H2N
NH2
–1e–
HN
Disadvantages
Results fade over time,
azide inhibits
enzyme activity
DAB
500 pg
Brown
Dry powder
Fast color development,
low background
More safety
precautions than for
other substrates
Purple
Liquid substrate;
High sensitivity, nonfading
Opti-4CN™ 100 pg
Opti-4CN kit
color, low background
Azide inhibits
enzyme activity
More expensive
than 4CN
Amplified
5 pg
Purple
Amplified Opti-4CN kit
Opti-4CN
More steps than
unamplified protocol
Best sensitivity available,
no extra materials (such as X-ray film) needed
Colorimetric AP BCIP/NBT
100 pg
Purple
Dry powder, liquid
Stable storage of data
substrate, Immun-Blot kits
Detects endogenous
phosphatase activity
Amplified AP
High sensitivity
Immun-Blot kit
More steps than
unamplified protocol
••
NH
H2N
+ HRP–O + H2O
•
HN+
H2N
NH2
O
Polymerization
to complex
brown precipitate
NH
Fig. 5.7. Colorimetric detection
options with HRP. DAB and 4CN
are commonly used chromogenic
substrates for HRP. In the presence
of H2O2, HRP catalyzes the
oxidation of the substrate into a
product that is visible on a blot.
Left, reaction with DAB; right,
reaction with 4CN.
H2O
NH2
–1e– Quinone iminium cation radical
n
H CI
Insoluble purple
product
BCIP
ONa
O
CI
P
ONa
O
Br
N
H
AP + H2O
– OPO32–
CI
NBT
O
Br
H
H
2
Advantages
CI
HRP + H2O2
•+
n Colorimetric AP systems — use soluble 5-bromo4-chloro-3-indolyl phosphate (BCIP) and nitroblue
tetrazolium (NBT) as substrates to produce a stable
reaction product that will not fade (Figures 5.8 and
5.9A). AP can easily be inactivated by exposure
to acidic solutions. Multiple probing of the same
membrane with alternative antibody probes can be
performed using substrates that produce different
colors, such as blue and red (Blake et al. 1984,
Turner 1983, Kurien 2003)
Colorimetric HRP 4CN
500 pg
Purple
Dry powder, liquid
Fast color development,
substrate, Immun-Blot® kits low cost, low background
Amplified
10 pg
Purple
BCIP/NBT
••
H2N
n
DetectionSignal
Sensitivity Color
Product Options
OH
HRP–O
Table 5.4. Colorimetric detection systems.
Detection
Method
Substrate
4CN
DAB
+
N
H
–O
N+
BC indoxyl intermediate
Br
CI
N
+
N
N
N
C
C
N
N
O
N+
N
O
N+ O–
O
O
O
H
N
N
H
O
Indigoid dye
(purple precipitate)
Br
CI
H
N N
N
–O
N+
O
N
C
C
N
N
O
O
Insoluble diformazan
(blue precipitate)
N
N
H
N+ O–
O
Fig. 5.8. AP colorimetric
development. In the colorimetric
system, AP catalyzes the substrates
BCIP and NBT to produce a
colored precipitate visualizing the
protein on a western blot. First the
dephosphorylation of BCIP by AP
occurs, yielding a bromochloro
indoxyl intermediate. The indoxyl is
then oxidized by NBT to produce
an indigoid dye (purple precipitate).
The NBT is also reduced by the
indoxyl, opening the tetrazole ring
to produce an insoluble
diformazan (blue precipitate). The
combination of the indigoid dye
of the BCIP and the insoluble
formazan of the NBT forms a
purple-blue colored precipitate.
37
Protein Blotting Guide
Theory and Products
Premixed and Individual Colorimetric Substrates
Table 5.5. Chemiluminescence detection systems.
A
Premixed enzyme substrate kits and development
reagents, including powdered 4CN and DAB color
development reagents, are also available. The premixed
kits are convenient and reliable, and they reduce
exposure to hazardous reagents used in the color
development of protein blots.
Immun-Blot Amplified AP Kit
TABLE OF CONTENTS
Increased sensitivity in western blot experiments can
be achieved by utilizing an amplified AP procedure
(Bayer and Wilchek 1980, Chaiet and Wolf 1964,
Guesdon et al. 1979, Hsu et al. 1981). This detection
system (Figure 5.10A) begins by using a biotinylated
secondary antibody. Relying on the specific binding
properties of biotin and avidin, a complex of streptavidin
and biotinylated AP is then added to the membrane.
Because streptavidin will bind more than one molecule
of biotin, the initial site of the primary antibody-to-antigen
binding is effectively converted into multiple AP binding
sites available for color development (Figure 5.10A).
Color development is performed using conventional AP
substrates, as discussed previously. The Immun-Blot
amplified AP kit increases the detection sensitivity of
colorimetric western blotting to ≥10 pg of protein.
Detection
Substrate
Sensitivity
Detection
Product Options
Advantages
Disadvantages
Chemiluminescent HRP
Immun-Star™ HRP
Luminol
Conjugates
Short (30 sec) exposure
Azide inhibits enzyme activity
HRP substrate
Signal duration of 6–8 hr
B
Immun-Blot kits
Compatible with PVDF
and nitrocellulose
Working solution stable for
24 hr at room temperature
Immun-Star™ WesternC™ Luminol
Signal duration up to 24 hr
Amplified Opti-4CN substrate and detection kits are
based on proprietary HRP-activated amplification
reagents from Bio-Rad. These kits allow colorimetric
detection to 5 pg, which is comparable to or greater
than the sensitivity achieved with radiometric or some
chemiluminescence systems but without the cost
or time involved in darkroom development of blots.
Femtogram
Optimized for CCD imagers
High sensitivity
Immun-Star AP
Fig. 5.9. Colorimetric and chemiluminescent blots. A dilution
of a GST fusion protein was immunodetected using a monoclonal
antibody specific to GST followed by A, an AP-conjugated secondary
antibody and BCIP/NBT substrate for colorimetric detection, or B, an
HRP-conjugated secondary antibody and Immun-Star™ WesternC™
chemiluminescent substrate for chemiluminescence detection.
A
S
A
Multiple APs are available
to convert substrate (S) to
colored precipitate (P)
D
S
S
P
Biotinylated
secondary antibody
binds to primary
antibody
Conjugates
30 sec to 5 min exposure
CDP-Star
10 pg
AP substrate
Signal duration up to 24 hr
Immun-Blot kits
Blot can be reactivated
be captured on X-ray film or by a charge-coupled
device (CCD) imager such as the ChemiDoc™ XRS+
and VersaDoc™ MP systems. This technology is easily
adapted to existing western blotting procedures
because chemiluminescence uses enzyme-conjugated
antibodies to activate the light signal. The blocking and
wash methods are familiar procedures.
The advantages of chemiluminescent western blotting
Pover other methods are its speed and sensitivity
(Table 5.5). This method is perfect for CCD imaging,
which avoids the slow film step. Exposure times with
P
Complex of streptavidin
and biotinylated-AP
binds to biotin of
secondary antibody.
Endogenous phosphatase
activity may lead to
false positives
average blots are usually 5 sec to 5 min, depending
on the sensitivity of the substrate. This is a large
improvement over 125I systems, which can require up
to 48 hr for film exposure. Detection of protein down to
femtogram amounts is possible with these systems.
This is more sensitive than most colorimetric systems
and approximately equal to radioisotopic detection.
The detection sensitivity depends on the affinity of
the protein, primary antibody, secondary antibody, and
HRP substrate, and can vary from one sample
to another.
Luminol
CDP-Star
O
NH2
O
O
O
CH3
CI
NH
OPO3Na2
NH
AP + H2O
CI
O
HRP + H2O2
B
B
AP converts
chemiluminescent
substrate (S), which
emits light
AP-conjugated
secondary antibody
binds to primary
antibody
E
S
– OPO32–
O
O
O
P
NH2
CH3
CI
Film or phosphor
screen exposed by
emitted light
O–
O–
N
O
O
N
Peroxy
intermediate
CI
O–
O
O
CI
O
CH3
*
O–
+
*
NH2
COO–
CI
Product in excited state
Fig.
detection
methods.
S
C 5.10. Colorimetric and chemiluminescence
F
A, Detection using Immun-Blot amplified AP kit; B, Detection using
Immun-Star™ chemiluminescent kit.
P
38
Azide inhibits enzyme activity
Chemiluminescent AP
Chemiluminescence Detection
Chemiluminescence is a chemical reaction in which a
chemical substrate is catalyzed by an enzyme, such
as AP or HRP, and produces light as a by-product
(Figures 5.6B, 5.9B, and 5.10B). The light signal can
Conjugates
Opti-4CN ™ and Amplified Opti-4CN Substrate and Detection Kits
Colorimetric HRP detection with 4CN presents very
low background and a detection sensitivity of about
500 pg of antigen. Bio-Rad’s Opti-4CN kit improves
this detection sensitivity to 100 pg. Opti-4CN is
available as a premixed substrate kit or combined
with an HRP-conjugated antibody in a detection kit.
1–3 pg
Immun-Blot ® Assay Kits
Immun-Blot assay kits provide the reagents required
for standard HRP/4CN or AP colorimetric detection on
western blots with the added convenience of premixed
buffers and enzyme substrates. In addition, these kits
contain a secondary antibody conjugated to either
HRP or AP. All kit components are individually tested
for quality in blotting applications. Included in each
kit is an instruction manual with a thoroughly tested
protocol and troubleshooting guide that simplifies
immunological detection.
Method
COO–
Light emission
Light emission
Product in
excited state
Fig. 5.11.
Chemiluminescence
detection. The
secondary antibody is
linked to an enzyme,
which catalyzes a
reaction leading
to light emission.
Left, CDP-Star or
another 1,2-dioxetane
AP substrate is
dephosphorylated
by AP, resulting in
formation of an anion
in an excited state
that emits light.
Right, luminol
oxidized by HRP in
the presence of H2O2
leads to the formation
of a 3-aminophthalate
dianion and the
release of light.
Links:
Immun-Blot AP
Colorimetric Kits
Immun-Blot Opti-4CN
Colorimetric Kits
Immun-Star HRP
Chemiluminescence Kits
Immun-Star AP
Chemiluminescence Kits
ChemiDoc XRS+ System
VersaDoc MP Systems
Au
39
Protein Blotting Guide
Theory and Products
Fluorescence Detection
In fluorescence, a high-energy photon (ℎnex ) excites a fluorophore,
causing it to leave the ground state (S0 ) and enter a higher energy state
(S'1). Some of this energy dissipates, allowing the fluorophore to enter a
relaxed excited state (S1). A photon of light is emitted (ℎnem ), returning the
fluorophore to the ground state. The emitted photon is of a lower energy
(longer wavelength) due to the dissipation of energy while in the excited
state.
S'1
S1
Energy
nex
nem
S0
When using fluorescence detection, consider the following optical
characteristics of the fluorophores to optimize the signal:
n
TABLE OF CONTENTS
n
n
n
Quantum yield — efficiency of photon emission after absorption of
a photon. Processes that return the fluorophore to the ground state
but do not result in the emission of a fluorescence photon lower the
quantum yield. Fluorophores with higher quantum yields are generally
brighter
Immun-Star™ Chemiluminescence Kits
Immun-Star kits include either CDP-Star substrate
(activated by AP) or luminol (activated by HRP) and
produce a strong signal on either nitrocellulose or PVDF.
The light signal generated with Immun-Star kits not only
gives a fast exposure but also lasts for as long as
24 hr (Immun-Star™ WesternC™ and Immun-Star AP
kits) after initial activation of the blot. These blots can
also be reactivated with fresh substrate, even weeks
after the signal has been depleted. They can also be
stripped and reprobed multiple times.
Stokes shift — difference in the maximum excitation and emission
wavelengths of a fluorophore. Since some energy is dissipated while
the fluorophore is in the excited state, emitted photons are of lower
energy (longer wavelength) than the light used for excitation. Larger
Stokes shifts minimize overlap between the excitation and emission
wavelengths, increasing the detected signal
Excitation and emission spectra — excitation spectra are plots of
the fluorescence intensity of a fluorophore over the range of excitation
wavelengths; emission spectra show the emission wavelengths of the
fluorescing molecule. Choose fluorophores that can be excited by the
light source in the imager and that have emission spectra that can
be captured by the instrument. When performing multiplex western
blots, choose fluorophores with minimally overlapping spectra to
avoid channel crosstalk
Relative intensity
Absorption
(excitation)
400
Emission
Spectral
overlap
500 600
Wavelength, nm
700
800
Several fluorophores spanning a wide range of
excitation and emission wavelengths are now
available, including some based on organic dyes
(for example, cyanine and fluorescein), nanocrystals
of semiconductor material (for example, Qdot
nanocrystals), and naturally fluorescent proteins (for
example, phycobiliproteins such as phycoerythrin and
allophycocyanin).
Fluorescence detection (Figure 5.13) offers several
advantages over other methods:
n
The Immun-Star WesternC chemiluminescence kit
is designed for use with Precision Plus Protein™
WesternC™ standards and CCD imaging systems. It
offers femtogram-level sensitivity and compatibility
with any HRP-conjugated antibody (Figure 5.12).
Strong signal intensity optimized for CCD imaging is
produced.
n
Extinction coefficient — measure of how well a fluorophore absorbs
light at a specific wavelength. Since absorbance depends on path
length and concentration (Beer’s Law), the extinction coefficient is
usually expressed in cm –1 M –1. As with quantum yield, fluorophores
with higher extinction coefficients are usually brighter
Stokes
shift
40
Safety is another advantage of chemiluminescence
detection. It does not have the disadvantages
related to isotope detection, such as exposure of
personnel to radiation, high disposal costs, and
environmental concerns.
n
Fig. 5.12. Detection of antigen and Precision Plus Protein WesternC
standards using the Immun-Star WesternC chemiluminescence
detection kit. Proteins and 5 μl standards (lane 1) and a dilution series
of an E. coli cell lysate (lanes 2–6) were electrophoresed on a 4–20%
Criterion™ gel and transferred to a nitrocellulose membrane. The blot was
probed with an antibody specific for GST fusion proteins followed by an
HRP-conjugated secondary antibody and StrepTactin-HRP conjugate.
After a 5 min incubation in the Immun-Star WesternC detection solution,
the blot was imaged on a ChemiDoc™ XRS+ imager for 5 sec.
Multiplexing — use of multiple and differently
colored fluorophores for simultaneous detection
of several target proteins on the same blot. When
detecting multiple proteins in a fluorescent multiplex
western blot, ensure the fluorescent signals
generated for each protein can be differentiated.
Use primary antibodies from different host species
(for example, mouse and rabbit) and secondary
antibodies that are cross-absorbed against other
species to avoid cross-reactivity. Use fluorophores
conjugated to secondary antibodies with distinct
spectra so they can be optically distinguished from
each other to avoid cross-channel fluorescence
A. Bioluminescence
B. Chemifluorescence
Substrate
Substrate
Product
Product
C. Autoradiography
D. Immunogold
Au
Dynamic range — a 10-fold greater dynamic range
over chemiluminescence detection and, therefore,
better linearity within detection limits
Stability — many fluorescent molecules are stable
for a long period of time, allowing blots to be
stored for imaging at a later date — often weeks
or months later — without significant signal loss.
Most fluorescence techniques are also compatible
with stripping and reprobing protocols (provided the
blots are not allowed to dry out between successive
western detection rounds)
Fig. 5.14. Mechanism of detection chemistries. In bioluminescence
detection (A), the enzyme reaction itself emits light, while in
chemifluorescence (B), the product of the reaction is fluorescence. In
autoradiography (C), the secondary antibody itself carries a radioactive
label, and in immunogold labeling (D), the secondary antibody is
labeled with gold and signal is enhanced by silver precipitation.
Other Detection Methods
Bioluminescence
Bioluminescence is the natural light emission by
many organisms. Bioluminescence systems differ in
the structure and function of enzymes and cofactors
involved in the process as well as the mechanism of
the light-generating reactions. Bioluminescence is also
used as a detection method for proteins and nucleic
acids on a membrane.
Fluorescence Detection
In fluorescence detection, a primary or secondary
antibody labeled with a fluorophore is used during
immunodetection. A light source excites the
fluorophore and the emitted fluorescent signal is
captured by a camera to produce the final image
(see sidebar at left).
A drawback of fluorescence detection is its reduced
sensitivity compared to chemiluminescence methods,
such that detection using low-affinity antibodies or
of low-abundance proteins may yield lower signals.
Photostable fluorophores, improved instrumentation,
and membranes with low autofluorescence
characteristics are available to allow fluorescence
detection to approach the sensitivities seen with
chemiluminescence techniques.
Fig. 5.13. Multiplex fluorescence detection of a two-fold dilution
series of two proteins, GST (red) and soybean trypsin inhibitor
(green). Starting concentration was 500 ng of each protein. Precision
Plus Protein™ WesternC™ standards were used as markers.
Bioluminescence detection involves incubation
of the membrane (with bound antigen-antibodyenzyme complex) in a bioluminogenic substrate and
simultaneous measurement of emitted light (Figure
5.14A). The substrate involved in this detection system
is a luciferin-based derivative. Light detection is
performed using a photon-counting camera and the
blotted proteins are visualized as bright spots.
This technique is similar to chemiluminescence in
its sensitivity and speed of detection, but it is not
widely used and few bioluminogenic substrates
Links
Immun-Star HRP
Chemiluminescence Kits
Immun-Star AP
Chemiluminescence Kits
Precision Plus Protein
Western C Standards
41
Protein Blotting Guide
Theory and Products
are commercially available. PVDF is the preferred
membrane for bioluminescence detection because
nitrocellulose membranes may contain substances
that inhibit luciferase activity.
A
B
Table 5.6. Comparison of western blot documentation and analysis methods.
Imaging System
Autoradiography
TABLE OF CONTENTS
The gamma-emitting radioisotope 125I can be used
to label lysines in immunoglobulins for radiometric
antigen detection (Figure 5.14C). Direct immunological
detection (using labeled secondary antibodies) of as
little as 1 pg of dotted immunoglobulin is possible with
high specific activity 125I probes. Radiolabeled blots
can be detected using X-ray film, a method known
as autoradiography. Due to the hazards associated
with radiolabeled conjugates, autoradiography is
declining in popularity in favor of colorimetric and
chemiluminescence methods.
Immunogold Labeling
Immunogold detection methods utilize gold-labeled
secondary antibodies for antigen detection. Because
this method has relatively low sensitivity and the
signal is not permanent, silver enhancement methods
similar to those described on page 32 for colloidal
gold total protein stains were developed as a means
of enhancing the signal (Figure 5.14D). With silver
enhancement, a stable dark brown signal with little
background is produced on the blot and sensitivity
is increased 10-fold, equivalent to colorimetric
AP detection and several times more sensitive
than autoradiography.
C
42
XR+
VersaDoc™
XRS+
MP 4000 MP 5000
Pharos™
FX
FX Plus
GS-800™
PMI
D
Total Protein Stain
•
•
•
•
— ——
•
Colorimetric —
Fluorescent
•
•
•
•
•
•
•
•
—
Stain-free
•
—— —
——
—
— —
­­
SupercooledSupercooled
Imager Type CCDCCD
CCD
CCDCCD
LaserLaserLaserDensitometer
Fig. 5.15. Stripping and reprobing PVDF membranes. E. coli lysate
containing human transferrin and a GST-tagged protein was loaded
on a gel and blotted onto PVDF membranes. A, the blot probed with
an anti-GST antibody and developed with Immun-Star™ WesternC™
chemiluminescent substrate. B, the same blot subsequently stripped
of antibody and reprobed with the secondary antibody and developed
with chemiluminescent substrate to demonstrate removal of primary
antibody. This blot was also reprobed with StrepTactin-HRP to
visualize the ladder. C, the stripped blot reprobed with an anti-human
transferrin antibody. D, a control blot that did not undergo the stripping
procedure probed with the anti-human transferrin antibody.
Blots can be stripped and reprobed several times
but each round of stripping removes some sample
from the blot. This decreases the sensitivity of later
rounds of detection and may necessitate longer
exposure times or more sensitive detection methods.
Excitation Type
•
•
•
•
•
— ——
N/A
Trans UV/Vis
•
•
•
•
— ——
N/A
Epi white
—
•
•
— ——
N/A
LED RGB
—
—
—
•
•
•N/A
Laser RGB
——
—
——
Imaging — Analysis and Documentation
Several methods are employed to document western
blotting results (Table 5.6).
n
n
When probing a blot multiple times:
n
n
n
Stripping and Reprobing
Membranes that have been detected with
noncolorimetric methods such as chemiluminescent
or fluorescent techniques can be stripped of
antibodies for use in subsequent rounds of Western
detection (Figure 5.15). This allows reuse of the same
blot for investigation of different proteins and saves
both time and sample material.
EZ
ChemiDoc™
Immunodetection
•
•
•
— ——
—
Chemiluminescence—
—
•
•
•
•
— ——
•
Colorimetric—
Fluorescence ——
—
•
•
•
•—
—
•—
Autoradiography
——
—
——
— •
•
•
•
•—
—
Chemifluorescence—
—
—
Chemifluorescence
Chemifluorescence is the enzymatic conversion of
a substrate to a fluorescent product (Figure 5.14B).
Fluorogenic compounds (nonfluorescent or weakly
fluorescent substances that can be converted to
fluorescent products) are available to use with a
wide variety of enzymes, including AP and HRP. The
enzyme cleaves a phosphate group from a fluorogenic
substrate to yield a highly fluorescent product. The
fluorescence can be detected using a fluorescence
imager such as the PharosFX™ or VersaDoc MP
system. Chemifluorescence can provide a stable
fluorescent reaction product so blots can be scanned
at a convenient time. The method is compatible with
standard stripping and reprobing procedures.
Gel Doc™
n
If detecting proteins of different abundance or
when using antibodies with very different binding
affinities, first detect the protein with the lower
expected signal sensitivity
Comparisons of target protein abundance
among different rounds of detection will be
unreliable, as some sample is removed during
the stripping process
If possible, use PVDF membranes. PVDF is more
durable and resists loss of sample better than
nitrocellulose membranes
After stripping a blot, test it for complete removal
of the antibody. If chemiluminescent detection
methods were used, confirm removal of the
secondary antibody by incubation with fresh
chemiluminescent substrate. Detect any remaining
primary antibodies by incubation with an
HRP-labeled secondary antibody followed by
incubation with fresh chemiluminescent substrate.
If any antibody is detected using these tests,
restrip the blot before subsequent experiments
n
n
Densitometers — based on high-performance
document scanners with minor modifications
(leak-resistant scanning surface, built-in calibration
tool) and utilize visible light for analysis of
electrophoresis gels (transmission mode) and blots
(reflective mode) stained with visible dyes
CCD (charge-coupled device) cameras — versatile
systems that image both gels and blots, and operate
with either trans-illumination provided by light
boxes (visible or UV) positioned underneath the gel
for imaging a variety of stains (Coomassie, silver,
fluorescence) or epi-illumination of blots detected
using colorimetric or fluorescence techniques.
Different illumination wavelengths are available for
multiplex fluorescence immunodetection. CCD
cameras can also be used without illumination
to detect luminescent signals. Supercooled CCD
cameras reduce image noise, allowing detection of
faint luminescent signals
These imagers can be used for autoradiographic
detection techniques
n
X-ray film — widely used for imaging
autoradiographic and chemiluminescent blots but
suffers from a limited dynamic range as well as a
nonlinear response through this range. This method
also requires the investment in and maintenance of a
film developer and often requires processing multiple
film sheets to obtain a usable image. Decreasing
costs, higher resolution, better ease of use, and a
larger, more linear dynamic range are making CCD
cameras and imagers preferred over film for these
detection techniques
Luminescence Detection
Laser-based imagers — offer the highest sensitivity,
resolution, and linear dynamic range and are
powerful image acquisition tools for blots stained
with fluorescent dyes such as SYPRO Ruby.
These imagers can be configured with lasers of
different wavelengths, allowing single- or multiplex
fluorescence immunoblot detection
For chemiluminescence detection, CCD imaging
is the easiest, most accurate, and rapid method.
Traditionally, the chemiluminescent signal from blots
was detected by X-ray film. Film is a sensitive medium
for capturing the chemiluminescent signal but suffers
from a sigmoidal response to light with a narrow region
or linear response, which limits its dynamic range.
To gather information from a blot that has both intense
and weak signals, multiple exposures are required
to produce data for all samples in the linear range of
the film. A process termed preflashing can improve
linearity but this requires extra equipment and effort.
Additionally, quantitation of data collected by exposure
to film requires digitization (that is, scanning of X-ray
film with a densitometer).
Phosphor imagers — laser-based systems capable
of imaging storage phosphor screens. These
screens have a large dynamic range and offer
excellent sensitivity and quantitative accuracy with
imaging times that are a fraction of those for film.
CCD cameras have a linear response over a broad
dynamic range — 2–5 orders of magnitude —
depending on the bit depth of the system. CCD
cameras also offer convenience by providing a digital
record of experiments for data analysis, sharing, and
Links:
Gel Doc EZ Imager
Gel Doc XR+ System
ChemiDoc XRS+ System
VersaDoc MP Systems
PharosFX Systems
GS-800 Calibrated
Densitometer
43
Protein Blotting Guide
Theory and Products
archiving, and by eliminating the need to continually
purchase consumables for film development. CCD
cameras also approach the limit of signal detection in
a relatively short time. For example, the VersaDoc™ MP
5000 imaging system can reach the limit of detection
of a given experiment in <1 min, compared to 30 min
required by Kodak Bio-Max film for the same experiment.
Digital Imaging for Fluorescence, Chemifluorescence,
and Colorimetric Detection
TABLE OF CONTENTS
Fluorescence, chemifluorescence, and colorimetric
detection all benefit from the advantages of digital
imaging: convenience, digital records of experiments,
sensitive limits of detection, and wide dynamic ranges.
Fluorescent and chemifluorescent signals can be
detected with a wide range of imaging systems,
including both CCD and laser-based technologies.
For example, the VersaDoc MP and Pharos™ FX Plus
systems can be used similarly to detect fluorescent
and chemifluorescent signals. The decision to use
one type of technology over another depends on
budget and requirements for limit of detection and
resolution. CCD systems are generally less expensive
than laser-based systems. While the dynamic range
of CCD imaging systems varies from 2 to 5 orders of
magnitude, laser-based systems typically provide a
wide dynamic range of 4.8 orders of magnitude. The
resolution of CCD and laser-based systems are similar,
with the finest resolution settings generally being
50 μm or less. Another advantage of fluorescence
and chemifluorescence detection is that CCD and
laser-based detection limits generally far exceed the
dynamic ranges of the fluorescence assays currently
used for protein detection.
film is nonlinear but it can easily be made linear by
preexposing the film to a flash of light. Phosphor
imagers, such as Bio-Rad’s PharosFX Plus or PMI™
system, offer an alternative to film detection methods.
The initial investment in instrumentation offers
increased sensitivity and dynamic range compared
to X-ray film, and exposure times are 10 to 20 times
shorter than those for film. The ability to accurately
quantitate data is also much greater with storage
phosphor screens because the linear dynamic range
of phosphor imagers is significantly greater — 5 orders
of magnitude — enabling accurate quantitation and
the elimination of overexposure and saturated signals.
bands in the image. The software can measure
total and average quantities and determine relative
and actual amounts of protein. Gel imaging software
is also capable of determining the presence/
absence and up/down regulation of bands, their
molecular weights, isoelectric points, and other
values. Signal intensities can be quantitated and
compared to determine relative signal and to
generate other data such as Rf values for molecular
weight determinations
n
Analysis Software
Blot detection using an imaging system needs a robust
software package for image acquisition. In addition,
a good software package can magnify, rotate, resize,
overlay, and annotate the corresponding gel and blot
images, allowing export of the images to common
documentation software. A good software package
also allows analysis of the blot image and comparisons
of relative signal intensities, protein molecular weight,
or other aspects.
n
Quantity One® 1-D analysis software — acquires,
quantitates, and analyzes a variety of data, including
radioactive, chemiluminescent, fluorescent, and
color-stained samples acquired from densitometers,
storage phosphor imagers, fluorescence imagers,
and gel documentation systems. The software
allows automatic configuration of these imaging
systems with appropriate filters, lasers, LEDs, and
other illumination sources and allows manual or
automated analysis of PAGE gels and western blots
PDQuest™ 2-D analysis software — used for 2-D gel
electrophoresis analysis
For automated acquisition and analysis of gel and blot
images, Bio-Rad offers:
n
Image Lab™ software (Figure 5.16) — control
software for a variety of Bio-Rad imaging systems,
Image Lab software automatically determines
the image with the best signal-to-noise ratio and
generates a report. It also provides sophisticated
algorithms to determine the number of lanes and
Colorimetric samples can be easily recorded and
analyzed with a densitometer such as the GS-800™
calibrated densitometer. The densitometer provides
a digital record of the blot, excellent resolution,
reproducible results, and accurate quantitation.
The GS-800 also uses red, green, and blue color
CCD technology to greatly improve the imaging of
a wide range of colorimetric detection reagents.
Autoradiography
To detect the commonly used radioisotopes, 35S,
32P, 33P, 12C, and 125I, the most widely used method
is autoradiography on X-ray film. Autoradiography
provides a good combination of sensitivity and
resolution without a large investment in detection
substrates or imaging systems. For direct
autoradiography without intensifying screens or
scintillators, the response of the film is linear only
within a range of 1–2 orders of magnitude. When
intensifying screens or fluorographic scintillators
are used to increase sensitivity, the response of the
44
Links:
Image Lab Software
Fig. 5.16. Image Lab software. E. coli lysate was separated and
activated on a 4–20% Criterion™ TGX Stain-Free™ gel and transferred
onto a PVDF membrane. The membrane was imaged on a
Gel Doc™ EZ system and analyzed using Image Lab 3.0 software.
Quantity One 1-D Analysis
Software
PDQuest 2-D Analysis
Software
45
Protein Blotting Guide
Methods
TABLE OF CONTENTS
PART 2
Methods
Part 2 presents general
protocols for transfers of
proteins to membranes,
total protein detection,
and immunodetection.
46
47
Protein Blotting Guide
Methods
Protocols
Protocols
Electrophoretic Transfers
Electrophoretic Transfers
Reagent and Materials Preparation
Tank Blotting Procedure Part I
Prepare the Gel and Membrane Sandwich
1
TIPS
1
Gel equilibration removes
contaminating electrophoresis
buffer salts. If not removed,
these salts increase the
conductivity of the transfer
buffer and the amount of heat
generated during transfer.
Equilibration also allows the
gel to adjust to its final size
prior to electrophoretic
transfer. Gels shrink or swell
to various degrees in the
transfer buffer depending on
the acrylamide percentage
and the buffer composition.
TABLE OF CONTENTS
Equilibration is not necessary
(i) when the same buffer is
used for both electrophoresis
and transfer (for example,
native gel transfers), or (ii)
when using rapid semi-dry
transfer systems such as the
Trans-Blot® Turbo™ system
(consult the user manual for
the system you are using).
Buffer
Membrane and
filter paper
Prepare transfer buffer.
Refer to Table 2.2 or
the instruction manual
for the transfer cell you
are using for the buffer
requirements of each
transfer unit. Add
200 ml of buffer per gel
for equilibration of gels
and transfer materials.
Foam pad
2
2
et a foam pad in
W
transfer buffer and place
it on the submerged side
of the cassette.
3
Wet a piece of filter
paper in transfer buffer
and place it on top of the
foam pad. Use a blot roller
to remove trapped air.
Membrane
Gel
Filter paper
Foam pad
4
Gel (rinsed first)
3
3
Equilibate (if necessary).
Wet and equilibrate
membranes in transfer
buffer for at least 5 min.
For PVDF membranes,
wet in 100% methanol
for ~1 min prior to
equilibration in transfer
buffer. Rinse gels in diH2O
and equilibrate in transfer
buffer for 15 min.
Images and material shown are based
on Bio-Rad tank blotting products.
Materials may differ based on your
blotting apparatus manufacturer.
48
pen a gel holder
O
cassette and submerge
the cathode (black) side
in transfer buffer.
Filter paper
2
Determine the proper
membrane for the specific
experiment. Select a
precut membrane and
filter papers or cut the
membrane and filter
paper to match the size
of the gel.
1
lace the equilibrated gel
P
on top of the filter paper. If
needed, gently use a blot
roller to remove trapped air.
5
lace the equilibrated
P
membrane on top of the
gel. Use a blot roller to
remove trapped air.
6
Wet a second piece of
filter paper in transfer
buffer and place it on top
of the membrane. Again,
roll to remove trapped air.
7
Soak a foam pad in
transfer buffer and place
it on top of the filter
paper, then close and
lock the cassette.
49
Protein Blotting Guide
Methods
Protocols
Protocols
Electrophoretic Transfers
Electrophoretic Transfers
Tank Blotting Procedure Part II
Semi-Dry Blotting Procedure
Assemble the Tank and Program the Power Supply
TIPS
Stirring during transfer helps
maintain uniform conductivity
and temperature. Failure
to properly control buffer
temperature may result in poor
transfer and poses a potential
safety hazard.
Electrophoretic transfer
entails large power loads and
consequently, heat generation.
The tanks are effective thermal
insulators and limit the efficient
dissipation of heat; thus, simply
placing the tank in the cold
room is not enough to remove
all of the heat generated
during transfer.
TABLE OF CONTENTS
Effective cooling required for
high-intensity field transfers
and recommended for long,
unsupervised runs can be
provided using the cooling coil
or Bio-Ice™ units included with
your transfer device.
Evaluate transfer efficiency
at various field strengths
(V/cm), staying within the
recommendations for
each instrument.
1
2
3
Add a stir bar and begin
stirring. If needed, begin
cooling the transfer tank with
an ice pack or cooling coil.
Insert the gel holder cassette
into the blotting module latch
side up, with the black side
of the cassette facing the
black side of the blotting
module. Repeat with additional
casettes if needed. Place
blotting module with cassettes
in the tank.
1
2
Safety cover
Cathode
assembly
Filter paper
4
Membrane
3
Filter paper
dd transfer buffer to the
A
tank until the buffer level
reaches the fill line.
6
lace the lid on top of the
P
cell, making sure that the
color-coded cables on the
lid are attached to the proper
electrode cards.
Connect the cables to the
power supply, making sure to
match the colors on the cables
to those on the power supply
inputs. Program the power
supply and start the run.
6
7
Note: Reversing field polarity by
switching cable colors will cause
irreversible damage to the electrodes.
7
50
pon completion of the run,
U
remove the cassettes and
disassemble the gel and
membrane sandwich.
emove the safety cover and
R
stainless-steel cathode assembly
and place the presoaked filter
paper (one sheet of extra thick or
three sheets of thick paper) onto
the platinum anode. Remove air
trapped between the paper and
the anode using a blot roller.
Carefully place the equilibrated
membrane on top of the filter
paper. Roll out any air trapped
between the transfer materials.
Anode
5
5
Soak the filter paper in transfer
buffer (two sheets of extra thick
or six sheets of thick filter paper).
Gel
4
For transfers using high power,
monitor the transfer carefully
and use cooling as needed.
Perform a test run to
determine the time required
for complete transfer. Times
may vary from 15 min to
overnight, depending on many
factors, including the power
setting, gel percentage, and
the size, shape, and charge
of the protein.
lace the transfer tank on
P
a magnetic stir plate and
fill the tank halfway with
transfer buffer.
8
ently place the equilibrated
G
gel on top of the membrane
and roll out trapped air.
TIPS
Evaluate transfer efficiency
at various field strengths
(V/cm), staying within the
recommendations for
each instrument.
Bio-Rad semi-dry systems
place the anode on the
bottom electrode. If using
a different system, consult
the owner’s manual for the
proper orientation of the gel
and membrane.
For transfers using high power,
monitor the transfer carefully
and use cooling as needed.
Perform a test run to
determine the time required
for complete transfer. Times
may vary from 15 min to 1 hr,
depending on many factors,
including the power setting,
and the size, shape, and
charge of the protein.
lace filter paper (one sheet
P
of extra thick or three sheets
of thick) onto the gel and roll
out trapped air.
Carefully place the cathode
assembly onto the transfer
stack and then place the safety
cover back onto the unit.
onnect the cables to the power
C
supply, making sure to match
the colors on the cables to those
on the power supply inputs.
Program the power supply (see
Chapter 4) and start the run.
Upon completion of the run,
remove the cathode assembly
and disassemble the gel and
membrane sandwich. If needed,
rinse the gel briefly with diH2O.
51
Protein Blotting Guide
Methods
Protocols
Protocols
Electrophoretic Transfers
Trans-Blot®
Turbo™
Microfiltration
Blotting Procedure
1
2
After gel electrophoresis, open the
transfer pack that matches your gel
(mini or midi) and place the anode stack
on the cassette base. Place single mini or
midi stacks in the middle of the cassette
base; two mini gels can be placed on a
midi stack with each gel bottom facing the
center. Use the blot roller to remove any air
trapped between the pad and membrane.
No equilibration is required.
lace the gel on the anode stack (which
P
includes the membrane) and the cathode
stack on the gel. Roll to remove trapped air.
1
Sample template with
attached sealing screws
Membrane
2
Sealing gasket
Gasket
support plate
3
Vacuum manifold
TABLE OF CONTENTS
3
4
5
6
52
lace the lid on the cassette and lock it
P
into place by turning the green knob
clockwise. Ensure the locking pins fully
engage their locking slots.
4
urn the instrument on and slide the
T
cassette into either cassette bay. If using
two cassettes, each must be using the
same size transfer pack.
5
tart the transfer. With the cassette
S
inserted into the instrument, press
TURBO and select the gel type. Press
A:RUN to start the top tray, B:RUN for
the bottom tray. Select LIST to select
a preprogrammed protocol or NEW to
create and run a new protocol.
t the end of the run, RUN COMPLETE
A
appears on the screen. Remove the
cassette from the instrument and unlock
the lid. (Caution: the cassette may be
warm.) Remove the membrane from
the transfer sandwich and discard the
remaining transfer pack materials.
6
Tubing and flow valve
7
repare the samples.
P
For best results, filter
or centrifuge samples
to remove particulate
matter that might restrict
the flow of solutions
through the membrane.
Assemble the unit as shown
in the illustration at left.
Adjust the flow valve so
the unit is exposed only
to atmospheric pressure.
Add samples. Remove any
air bubbles trapped in the
wells by gently pipetting the
solution up and down.
or best sample binding,
F
the entire sample should
be filtered by gravity flow
without vacuuming.
The membrane may be
washed by adding a volume
of buffer equal to the sample
volume in each well.
After application of
sample, the membrane
may be blocked and then
probed for the protein of
interest. Refer to the product
instruction manual for
detailed instructions.
TIPS
Application of the Vacuum
During the assay, do not leave
the unit with the vacuum
on. This may dehydrate the
membrane and may cause
halos around the wells.
Flow Valve
Proper positioning of the
flow valve relative to the
level of the apparatus is
important for proper drainage.
The speed of drainage is
determined by the difference in
hydrostatic pressure between
the fluid in the sample wells
and the opening of the flow
valve that is exposed to the
atmosphere. When the flow
valve is positioned below the
sample wells, proper drainage
may be achieved.
If a prolonged or overnight
incubation is desired, adjust
the flow valve so that the
vacuum manifold is closed
off from both the vacuum
source and atmosphere before
applying the samples. In this
configuration, solutions will
remain in the sample wells with
less than a 10% loss of volume
during extended incubations.
To apply a gentle vacuum
to the apparatus, adjust the
flow valve so that it is open to
the atmosphere, the vacuum
source, and the vacuum
manifold while the vacuum
is on. Then, use a finger to
cover the valve port that is
exposed to the atmosphere.
The pressure of your finger
on the valve will regulate the
amount of vacuum reaching
the manifold.
To remove the membrane,
leave the vacuum on while
loosening the screws
and removing the sample
template. Then turn off
the vacuum and remove
the membrane.
53
Protein Blotting Guide
Methods
Protocols
Protocols
Blot Stripping and Reprobing
Total Protein Detection
Based on Legocki and Verma 1981
General protocols are described below. For more details, refer to the instruction manual for the stain you are using.
1
TIPS
This protocol (based on
Legocki and Verma 1981)
uses low pH to gently
remove antibody from the
membrane. The protocol
removes little of the sample
proteins but may not remove
all antibodies with high
affinities for their targets.
SYPRO Ruby Stain
Consult the SYPRO Ruby Protein Stains
Instruction Manual (bulletin 4006173) for complete
instructions. Membranes stained with SYPRO
Ruby protein blot stain are best preserved by
allowing membranes to air dry.
1
2
TABLE OF CONTENTS
10 min x 2
3
5 min x 3
4
5
54
Prepare acidic glycine
stripping buffer (0.1 M
glycine, 20 mM magnesium
acetate, 50 mM KCl, pH 2.2;
for recipe, see page 60).
dd enough acidic
A
glycine stripping buffer
to completely cover the
developed membrane
and incubate at room
temperature for 10 min
with gentle agitation.
Repeat step 2 with
fresh acidic glycine
stripping buffer.
ash the blot three
W
times in TTBS for 5 min
each with gentle agitation.
Test for complete removal
of primary antibody by
reprobing with only the
secondary antibody and
redeveloping. No signal
should be detectable.
Re-block the membrane
and proceed to the next
detection protocol.
2
Pretreat — after electroblotting
proteins to a nitrocellulose membrane,
completely immerse the membrane in
7% acetic acid and 10% methanol,
and incubate for 15 min.
Wash — wash the membrane in
diH2O four times for 5 min each.
Ponceau S Stain
1
2
Stain — incubate the membrane
for 1–2 min in the staining solution.
Destain — destain the membrane
in water until the background clears.
Ponceau S staining is reversible and
will disappear after extended destains
in diH2O.
3
Wash — rinse the membrane in TBS
or deionized H2O before drying.
4
Proceed with immunodetection.
3
4
Stain — completely immerse the
membrane in SYPRO Ruby protein
blot stain for 15 min.
Wash — wash the membrane in diH2O
four to six times for 1 min each. Monitor
the membrane periodically using UV
epi-illumination to determine the level of
background fluorescence.
5
Image — image using epi UV or
green excitation.
6
Proceed to immunodetection
(if needed).
Colloidal Gold Total Protein Stain
Consult the Bio-Rad enhanced colloidal gold total
protein detection kit manual for complete instructions.
1
2
3
Wash — following transfer or protein
application, wash the membrane
three times for 20 min in high-Tween TBS
(TTBS with 0.3% Tween 20).
Water wash — wash the membrane
for 2 min in deionized H2O to remove
interfering buffer salts.
Stain — incubate the membrane with
colloidal gold stain, completely covering
the blot. Incubation times will vary with
the concentration of protein present
on the membrane. Most bands will be
visible in 1–2 hr. If increased sensitivity
is required, continue the assay using
the gold enhancement procedure.
55
Protein Blotting Guide
Methods
Protocols
Protocols
Immunodetection
Immunodetection
1
Wash — following transfer
or protein application, wash
the membrane for 5–10 min
in TBS.
3–6x
5–10 min
7
5–10 min
2
1 hr
3
Block — incubate the
membrane for 1 hr in
blocking solution.
8
1x
Wash — wash the
membrane twice in TTBS,
5–10 min per wash.
9
2x
5–10 min
TABLE OF CONTENTS
4
1–2 hr
2–6x
5–10 min
1 hr
5
6
Primary antibody —
dilute the antibody in
antibody dilution or
blocking solution (refer
to the instructions for
the antibody for the
recommended final
concentration). Incubate the
membrane for 1–2 hr in the
primary antibody solution
with gentle agitation.
Wash — wash the
membrane 2–6 times in
TTBS, 5–10 min per wash.
Antibody conjugate —
dilute the conjugate in
TTBS (refer to the
instructions for the
conjugate for the
recommended final
concentration). Incubate the
membrane for 1 hr in the
enzyme conjugate solution
with gentle agitation.
5–30 min
10
Wash — wash the
membrane 3–6 times in
TTBS, 5–10 min per wash.
Final wash — wash the
membrane in TBS to
remove the Tween 20 from
the membrane surface
prior to blot development
and imaging.
Note for Protein G-HRP Detection
Follow steps 1–8 of the immunodetection assay, except
use more stringent washes (steps 5 and 7). Wash the
membrane six times for 10 min each at these steps,
with strong agitation and a large volume of buffer to
reduce background. Then follow below for step 9:
Follow steps 1–4 on page 56. For step 5 (wash), use
TCBS instead of TTBS and then continue with steps 6–10.
Signal development — for
colorimetric development,
add detection substrate
and incubate for 5–30
min depending on the
specific reagents used.
For chemiluminescence/
fluorescence, see
next page.
Image, dry, and store —
image the blot on a
CCD laser-based imager,
or expose to X-ray film or
instant photographic film.
Develop the film according
to the manufacturer’s
instructions.
Notes for Multiplex Detection
Primary antibodies must be from different host species in
order to be detected in separate channels (for example,
mouse and rabbit).
Test primary antibodies individually to determine the
banding pattern for each on the membrane prior to
multiplexing. Once known, these antibodies can be diluted
and incubated simultaneously.
Secondary antibody conjugates should possess specificity
to the primary antibody host species (for example, goat
anti-mouse and goat anti-rabbit).
Secondary antibody conjugates should be highly crossabsorbed against other species to minimize cross-reactivity.
56
Notes for Chemiluminescence Detection
A
Place the membrane protein-side up on
a clean piece of plastic wrap or a plastic
sheet protector.
B
Add chemiluminescent substrate
solution. Use at least 0.1 ml per cm2 of
membrane (about 6 ml for a standard
7 x 8.5 cm gel).
C
Incubate the membrane for 3–5 min in
the chemiluminescent substrate solution.
D
Drain excess liquid from the blot
and seal the membrane in a bag or
sheet protector.
E
Notes for Amplified Opti-4CN™ Detection
Follow steps 1–8 of the immunological assay on
page 56. Then:
A
Incubate the membrane in diluted BAR
for 10 min.
B
Wash the membrane 2–4 times in 20%
DMSO/PBST for 5 min each time.
C
Wash 1–2 times in PBST for 5 min.
each time.
D
Incubate the membrane and diluted
streptavidin-HRP for 30 min.
E
ash the membrane twice in PBST for
W
5 min each time.
TIPS
If kept wet, blots using
HRP or AP conjugates can
be stored for several days
prior to development and
imaging. Leave blot in TBS,
or place membrane between
two pieces of filter paper
soaked in TBS, and place
in a sealable container.
Image the blot on a CCD imager such
as a ChemiDoc™ or VersaDoc™ system, or
expose to X-ray film (for example, Kodak
Continue with steps 9–10.
XAR or BioMax) or instant photographic
film, such as Polaroid Type 667 or 612.
Typical exposure times are 30 sec to 5
Notes for Amplified AP Detection
min. Develop the film according to the
Follow steps 1–5 of the immunodetection assay on
manufacturer’s instructions.
page 56. Then:
F
Notes for Fluorescence Detection
Follow steps 1–8 of the immunodetection assay.
Imaging of most fluorescent dye conjugates (Cy,
Dylight, Alexa Fluor, and IRDye dyes) can be performed
on wet or dry membranes. Imaging of fluorescent
protein conjugates (phycoerythrin, allophycocyanin)
should be performed on wet membranes for maximum
sensitivity. Refer to the table below for recommended
imager settings. Excitation and emission wavelengths
are similar for non-Bio-Rad imagers as well.
Red excitation
(e.g., Alexa 647, Cy5, DyLight 649)
Blue excitation
(e.g., FITC, Alexa 488,
DyLight 488)
Green excitation
(e.g., Alexa 555,
Cy3, DyLight 548,
TAMRA)
VersaDoc MP 695 BP
635 Ex/695 BP
PharosFX™
530 BP
488 Ex/530 BP
605 BP
532 Ex/605 BP
A
Incubate the membrane for 1–2 hr in
biotinylated secondary antibody solution.
B
While the blot is incubating in the
biotinylated antibody solution, prepare
the streptavidin-biotinylated AP
complex. Allow the complex to form
for 1 hr at room temperature.
C
ash the membrane twice in TTBS,
W
5–10 min per wash.
D
Incubate the membrane for 1–2 hr in the
streptavidin complex solution.
E
Continue with steps 7–10.
57
Protein Blotting Guide
Methods
Transfer Buffer Formulations
The following buffers are recommended for use with
all of Bio-Rad’s electrophoretic transfer cells. Care
should be taken when preparing these buffers because
incorrect formulation can result in a current that
exceeds the recommended conditions.
Use only high-quality, analytical grade
methanol. Impure methanol can increase transfer
buffer conductivity and yield a poor transfer.
In many cases, ethanol can be substituted
for methanol in the transfer buffer with minimal
impact on transfer efficiency. Check this using
your samples.
Do not reuse transfer buffer since the buffer
will likely lose its ability to maintain a stable pH
during transfer.
Do not dilute transfer buffers below their
recommended levels since this decreases their
buffering capacity.
TABLE OF CONTENTS
Do not adjust the pH of transfer buffers unless
specifically indicated. Adjusting the pH of transfer
buffers can result in increased buffer conductivity,
manifested by higher initial current output and
decreased resistance.
Increasing SDS in the transfer buffer increases
protein transfer from the gel but decreases binding
of the protein to nitrocellulose membrane. PVDF
membrane can be substituted for nitrocellulose
when SDS is used in the transfer buffer.
Addition of SDS increases the relative current,
power, and heating during transfer, and may
also affect antigenicity of some proteins.
Increasing methanol in the transfer buffer
decreases protein transfer from the gel and
increases binding of the protein to
nitrocellulose membrane.
25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH
8.3) (catalog #161-0734, without methanol, 1 L, 10x)
3.03 g
14.4 g
500 ml
200 ml
Adjust volume to 1 L with diH2O.
The pH will range from pH 8.1 to 8.5 depending on the
quality of the Tris, glycine, methanol, and diH2O.
58
TTBS wash solution, 1 L
25 mM Tris, 192 mM glycine, 20% methanol (v/v),
0.025–0.1% SDS (pH 8.3)
20 mM Tris-HCl, 500 mM NaCl, 0.05% Tween 20
(pH 7.5)
Add 2.5 to 10 ml 10% SDS to 1 L buffer prepared
above.
0.5 ml Tween 20
1 L TBS
Bjerrum Schafer-Nielsen Buffer, 1 L
Citrate-buffered saline (CBS)
48 mM Tris, 39 mM glycine, 20% methanol (pH 9.2)
Tris base Glycine diH2O
Methanol
5.82 g
2.93 g
500 ml
200 ml
Adjust volume to 1 L with diH2O.
Bjerrum Schafer-Nielsen Buffer with SDS, 1 L
48 mM Tris, 39 mM glycine, 20% methanol, 1.3 mM
SDS (pH 9.2)
Add 0.0375 g SDS (or 3.75 ml 10% SDS) to 1 L buffer
prepared above.
CAPS Buffer, 1 L
10 mM 3-(cyclohexylamino)-1-propanesulfonic acid,
10% methanol (pH 11.0)
CAPS diH2O
Methanol
2.21 g
500 ml
100 ml
Adjust volume to 1 L with diH2O.
Measure the pH and adjust as needed with NaOH.
Dunn Carbonate Buffer, 1 L
10 mM NaHCO3, 3 mM NaCO3,
20% methanol (pH 9.9)
NaHCO3 NaCO3 (anhydrous) diH2O
Methanol
0.84 g
0.318 g
500 ml
200 ml
Adjust volume to 1 L with diH2O.
0.7% Acetic Acid
Add 7 ml glacial acetic acid to 993 ml diH2O.
Detection Buffer Formulations
Towbin Buffer, 1 L
Tris base Glycine diH2O
Methanol
Towbin Buffer with SDS, 1 L
General Detection Buffers
Tris-buffered saline (TBS), 2 L
20 mM Tris-HCl, 500 mM NaCl (pH 7.5)
(catalog #170-6435, 1 L, 10x)
Tris base NaCl diH2O
4.84 g
58.48 g
1.5 L
Adjust pH to 7.5 with HCl.
Adjust volume to 2 L with diH2O.
20 mM citrate, 500 mM NaCl (pH 5.5)
Included in Immun-Blot® protein G kits.
TCBS wash solution, 1 L
20 mM citrate, 500 mM NaCl, 0.05% Tween 20 (pH 5.5)
0.5 ml Tween 20
1 L CBS
Blocking solution, 100 ml
3% gelatin-TBS
Add 3.0 g gelatin to 100 ml TBS.
Heat to 50°C; stir to dissolve.
or
3% BSA-TBS
Add 1.0 g BSA to 100 ml TBS; stir to dissolve.
or
5% nonfat milk-TBS
Add 5.0 g nonfat dry milk to 100 ml TBS;
stir to dissolve.
Note: Gelatin can clog membranes and cut off the
vacuum flow of microfiltration units; use an alternative
blocking solution with the Bio-Dot® or Bio-Dot SF
apparatus.
Note: Nonfat milk is not recommended for avidin/biotin
systems as milk contains endogenous biotin and may
cross-react with avidin-containing components in the
detection system.
Antibody dilution buffer, 200 ml
1% gelatin-TTBS
Add 2.0 g gelatin to 200 ml TTBS.
Heat to 50°C; stir to dissolve.
or
3% BSA-TTBS
Add 6.0 g BSA to 200 ml TTBS; stir to dissolve.
or
5% nonfat milk-TTBS
Add 10.0 g nonfat dry milk to 200 ml TTBS;
stir to dissolve.
Note: Gelatin can clog membranes and cut off the
vacuum flow of microfiltration units; use an alternative
blocking solution with the Bio-Dot or Bio-Dot SF
apparatus.
Note: Nonfat milk is not recommended for avidin/biotin
systems as milk contains endogenous biotin and may
cross-react with avidin-containing components in the
detection system.
Antibody buffer (for chemiluminescence, ImmunStar™ AP only)
0.2% nonfat milk-TTBS
Add 0.4 g nonfat milk to 200 ml TTBS; stir to dissolve.
Antibody buffer for protein G-HRP, 100 ml
1% gelatin-TCBS
Add 1.0 g gelatin to 100 ml TCBS.
Heat to 50°C; stir to dissolve.
Protein G-HRP conjugate solution, 100 ml
Mix 33 μl protein G conjugate solution in 100 ml 1%
gelatin in TCBS.
Streptavidin-biotinylated AP complex, 100 ml
33 μl streptavidin
100 ml TTBS
33 μl biotinylated AP
Incubate the complex 1–3 hr at room temperature
before use.
Total Protein Staining Buffers and Solutions
Amido black staining solution, 1 L
For nitrocellulose:
Amido black Methanol 5g
400 ml
Adjust volume to 1 L with diH2O.
or
Amido black Isopropanol
Acetic acid
5g
250 ml
100 ml
Adjust volume to 1 L with diH2O.
For PVDF:
Amido black Methanol Acetic acid
1g
400 ml
100 ml
Adjust volume to 1 L with diH2O.
Amido black destain solution, 1 L
For nitrocellulose:
Isopropanol
Acetic acid
250 ml
100 ml
Adjust volume to 1 L with diH2O.
For PVDF:
Methanol Acetic acid
400 ml
100 ml
Adjust volume to 1 L with diH2O.
59
Protein Blotting Guide
Methods
Coomassie Blue R-250 staining solution, 1 L
AP Substrate Buffers
Coomassie Blue R-250
Methanol
Acetic acid
AP color
development buffer
MgCl2 Tris base
diH2O
Adjust pH to 9.5 with HCl;
adjust volume to 1 L with diH2O.
1g
400 ml
100 ml
Adjust volume to 1 L with diH2O.
Coomassie Blue R-250 destaining solution, 1 L
Methanol
Acetic acid
5-bromo-4-chloroindolyl phosphate/nitroblue
tetrazolium (BCIP/NBT)
Dimethylformamide 0.7 ml
diH2O
0.3 ml
NBT30 mg
400 ml
100 ml
Adjust volume to 1 L with diH2O.
Ponceau S staining solution
Ponceau S
Trichloracetic acid (TCA) Sulfosalicylic acid diH2O
2g
30 g
30 g
80 ml
1% acetic acid or PBS
SYPRO Ruby blot pretreatment solution
70 ml
100 ml
830 ml
TABLE OF CONTENTS
Use TTBS wash solution (see page 59).
Substrate Buffers and Solutions
HRP Substrate Buffers
4-(chloro-1-naphthol) 4CN 60 mg
Methanol
20 ml
Protect mixture from light
3% H2O2
Substrate solution
600 μl
100 ml
Mix the two solutions together.
Use immediately. Alternatively,
use HRP conjugate substrate
solution in kit format.
For nitrocellulose
Add 500 μl enhancer
membrane blots:reagent to 10 ml Immun-Star
chemiluminescent substrate.
Store at 4°C for up to 1 week.
For PVDF
Immun-Star AP generates a
membrane blots: very fast light signal on PVDF
membrane; therefore, the use
of an enhancer is not
necessary. The substrate is
provided ready to use.
Immun-Star HRP substrate solution (kit format)
For nitrocellulose and PVDF
membrane blots:
A 1:1 mixture of luminol/
enhancer to peroxide buffer
is recommended. Use 10 ml per 100 cm2 of membrane (12 ml
for one 8.5 x 13.5 cm
Criterion™ blot).
HRP conjugate substrate solution
Dissolve contents of premixed
color development buffer in
diH2O to 1 L
Color reagent B
600 μl
Development buffer 100 ml
HRP color reagent A 20 ml
Use immediately.
Acidic glycine stripping buffer
Diaminobenzidine (DAB)
DAB
50 mg
TBS100 ml
3% H2O2
100 μl
Use immediately.
Glycine
Mg(CH3COO)2·4H2O
KCl
diH2O
Stripping Buffer
7.5 g
4.3 g
3.7 g
800 ml
Adjust pH to 2.2 with HCl.
diH2O
60
1 ml
15 mg
Immun-Star™ AP substrate solution (kit format)
Use 5 ml chemiluminescent
substrate per 100 cm2.
Colloidal gold blot staining solution
Dimethylformamide
BCIP
Add both solutions to 100 ml AP
color development buffer. Use
immediately. Alternatively, use
AP conjugate substrate solution
in kit format.
Ponceau S destaining solution
Acetic acid
Methanol
diH2O
0.233 g
12.1 g
800 ml
to 1 L
61
Protein Blotting Guide
Troubleshooting
TABLE
CONTENTS
TABLE
OFOF
CONTENTS
PART 3
Troubleshooting
The protocols included in this guide
are general recommendations for
transferring and detecting proteins on
blots. To help you optimize your protein
blotting results, this chapter includes
troubleshooting tips to improve both
transfer and detection.
62
63
Protein Blotting Guide
Troubleshooting
Transfer
Electrophoretic Transfer
ProblemCause
Solution
Poor electrophoretic transfer; bands Power conditions were inadequate or
•Increase the transfer time (thicker gels require
appear weak on blot (ensure proteins transfer time too short longer transfer times)
have been transferred by staining both the • Check the current at the beginning of the
gel and blot with a total stain. For example, run; it may be too low for a particular voltage
stain the gel with Bio-Safe™ Coomassie setting, indicating incorrect buffer composition.
or SYPRO Ruby stain, and stain the blot See the power guidelines for specific
with Ponceau S stain). Alternatively, one applications in Chapter 4
could use stain-free technology and • Use high-intensity blotting
PVDF membranes
• Use a power supply with a high current limit. If
an incorrect power supply is used, it is possible
to not reach the set voltage if the current of the
power supply is at its maximum limit
Power conditions were too high or transfer • Shorten transfer time
time too long (proteins may transfer through • Reduce transfer voltage
the membrane and into the filter paper)
• See “Overall poor binding to the membrane”
on page 65 for hints on how to improve binding
Transfer buffer was incorrect or
• Prepare fresh transfer buffer (never reuse
prepared incorrectly transfer buffer)
Proteins moved in the wrong direction • Check the gel/membrane sandwich assembly
(the gel/membrane sandwich may have • Check the assembly of the transfer cell
been assembled in the wrong order, the • Check the polarity of the connections
cassette inserted in the tank in the wrong to the power supply
orientation, or polarity of the connections
may be incorrect)
TABLE OF CONTENTS
The charge-to-mass ratio is incorrect
• Use a more basic or acidic transfer buffer to
(native transfers) increase protein mobility. A protein near its
isoelectric point (pI) will transfer poorly
(buffer pH should be 2 pH units higher or
lower than the pI of the protein of interest for
optimal transfer efficiency)
Protein precipitated in the gel
• Use SDS in the transfer buffer. SDS can
increase transfer efficiency but it can also
reduce binding efficiency to nitrocellulose and
affect reactivity of some proteins with antibodies
• Reduce or eliminate the alcohol in the
transfer buffer
The power supply circuit is inoperative or • Check the fuse
an inappropriate power supply was used
• Make sure the voltage and current output of
the power supply match the needs of the
blotting instrument
• Check the output capacity of the power supply
The gel percentage was too high • Reduce %T (total monomer) or %C (crosslinker).
(decreasing %T or %C increases gel pore Using 5%C (with bis-acrylamide as the
size and increases transfer efficiency) crosslinker) produces the smallest pore size
Regions of poor protein binding on the blot The membrane was not uniformly wet
• Ensure that membranes are uniformly wet
before transfer before transfer
• Because of the hydrophobic nature of PVDF,
the membrane must be completely soaked in
methanol prior to equilibration in aqueous
transfer buffer. A completely wet PVDF
membrane has a gray, translucent appearance
Buffer tank not filled to correct level
• Completely fill transfer tank with buffer. Transfer
tank must contain sufficient buffer to entirely
cover blot area
Swirls or missing bands; bands appear
Contact between the membrane and the • Carefully move the roller over the membrane in
diffuse on the blot
gel was poor; air bubbles or excess buffer both directions until air bubbles or excess buffer
remain between the blot and gel are removed from between gel and membrane
and complete contact is established
• Use thicker filter paper in the gel/membrane
sandwich
• Replace the foam pads. Pads compress and
degrade with time and will not hold the
membrane to the gel
64
ProblemCause
Solution
White spots on membrane
The membrane was not properly wetted • White spots on the nitrocellulose membrane
or had dried out indicate dry areas where protein will not bind.
If wetting does not occur immediately by
immersion of the sheet in transfer buffer, heat
distilled water until just under the boiling point
and soak the membrane until completely wet.
Equilibrate in transfer buffer until ready for use
• White spots on the PVDF membrane indicate
areas where the membrane was either
improperly prewetted or allowed to dry out.
Because of the hydrophobic nature of PVDF,
the membrane must be prewet in methanol prior
to equilibration in aqueous transfer buffer. Once
wet, do not allow membrane to dry out. If the
membrane dries, rewet in methanol and
re-equilibrate in TTBS (this may adversely effect downstream detection processes)
Broad or misshapen bands
Poor gel electrophoresis
• Artifacts of electrophoresis may occur as a
result of poor gel polymerization, inappropriate
running conditions, contaminated buffers, sample overload, etc. Consult your manual for
more details
Gel cassette pattern transferred to blot
Foam pads are contaminated or too thin • Clean or replace the foam pads
Excessive amounts of protein were loaded • Reduce the amount of protein on the gel
on the gel or too much SDS was used in • Reduce the amount of SDS in the transfer buffer
the transfer buffer. Proteins can pass • Add a second sheet of membrane to bind
through the membrane without binding excess protein
and recirculate through tank blotting systems
The transfer buffer was contaminated
• Prepare fresh transfer buffer
Overall poor binding to the membrane
Methanol in the transfer buffer is • Reduce the amount of methanol. This may
restricting elution improve transfer efficiency of proteins from
the gel but it also may decrease binding to
nitrocellulose membranes; 20% methanol is
generally optimal for protein binding
SDS in the transfer buffer reduces the
binding efficiency of proteins
• Reduce or eliminate SDS from the transfer buffer
Proteins passed through the membrane. • Use PVDF or 0.2 μm nitrocellulose
Proteins <15 kD may show decreased (smaller pore size)
binding to 0.45 μm membranes
• Decrease the voltage if using the
high-intensity option
• Place an additional membrane in the gel
sandwich to detect proteins that are being
transferred through the membrane
Microfiltration
ProblemCause
Solution
Leakage or cross-well contamination
The instrument was assembled incorrectly
• Retighten the screws under vacuum following
initial assembly to form a proper seal
The membrane was not rehydrated • Rehydrate the membrane prior to
after assembly loading samples
• Apply vacuum only until solutions are
removed from the sample wells, then
disconnect the vacuum
Uneven or no filtration
The membrane became clogged • Centrifuge samples or filter solutions prior to
with particulates application to remove particulates
The flow valve was positioned higher
• Position the flow valve lower than the level of
than the apparatus the sample wells or drainage will not occur
Bubbles obstructed the flow of liquid
• Use a needle to carefully break any bubbles,
being careful not to puncture the membrane
• Pipet liquid up and down to dislodge the bubbles
Improper blocking or antibody buffers
• Gelatin clogs the membrane; substitute BSA or
were used Tween 20 for gelatin in the detection procedure
Fluid pressure was not uniform
• Seal off unused wells or add solution to
unused wells
65
Protein Blotting Guide
Troubleshooting
ProblemCause
Solution
Halos around the wells
The membrane was not rehydrated • Rehydrate the membrane prior to loading samples
after assembly
• Apply vacuum only until solutions are
removed from the sample wells, then
disconnect the vacuum
Too much protein was loaded, overloading
• Determine optimum loading conditions by
the capacity of the membrane analyzing serial dilutions of samples
The blocking step was too short
• Use a blocking step that is as long as the
longest incubation period
Loading volume was too low
• The meniscus contacted the center of the well,
causing uneven distribution of protein sample.
The minimum loading volume is 100 μl
Detection
Immunodetection
ProblemCause
Solution
Overall high background
• Increase the concentration of blocker
• Increase the duration of the blocking step
• Use a different blocking agent
Blocking was incomplete
Blocker was impure. NFDM is not pure.
• Use a pure protein such as BSA or casein
The blocker may be contaminated as a blocker
with material that nonspecifically
binds probes
TABLE OF CONTENTS
Wash protocols were insufficient
• Increase the number, duration, or stringency
of the washes
• Include progressively stronger detergents
in the washes; for example, SDS is stronger
than Nonidet P-40 (NP-40), which is stronger
than Tween 20
• Include Tween 20 in the antibody dilution
buffers to reduce nonspecific binding
The blot was left in the enzyme substrate
• Remove the blot from the substrate solution
too long (colorimetric detection) when the signal-to-noise level is acceptable,
and immerse in diH2O
Contamination occurred during • Discard and prepare fresh gels and
electrophoresis or transfer transfer solutions
• Replace or thoroughly clean contaminated
foam pads if a tank blotter was used
Excessive amounts of protein were loaded • Reduce the amount of protein on the gel
on the gel or too much SDS was used in or SDS in the transfer buffer
the transfer buffer. Proteins can pass • Add a second sheet of membrane to
through the membrane without binding and bind excess protein
recirculate through a tank blotting system
The primary or secondary antibody was
• Increase antibody dilutions
too concentrated
• Perform a dot-blot experiment to optimize
working antibody concentration
Incubation trays were contaminated
• Clean the trays or use disposable trays
Nonspecific reactions between bound
proteins and probes
The primary or secondary antibody is • Use purified IgG primary antibody fractions and
contaminated with nonspecific IgG or with affinity-purified blotting-grade cross-adsorbed
IgG cross-reactive among species secondary antibody
Monoclonal antibodies reacted • Compare the binding of other monoclonal or
nonspecifically with SDS-denatured proteins polyclonal antibodies
• Blot native proteins as a comparison
Nonspecific interactions are occurring • Increase the ionic strength of the
due to ionic associations. For example, incubation buffers
avidin, a glycosylated protein, may bind to • Increase the number, duration, or stringency
more acidic proteins on blots of the washes
• Include progressively stronger detergents
in the washes; for example, SDS is stronger
than Nonidet P-40 (NP-40), which is stronger
than Tween 20
• Include Tween 20 in the antibody dilution
buffers to reduce nonspecific binding
66
ProblemCause
Solution
No reaction or weak signal
• Increase the amount of protein applied
• Concentrate the sample prior to loading
The sample load was insufficient
The detection system is not working or •Use a more sensitive assay system
is not sensitive enough
•Include proper positive and negative control
antigen lanes to test for system sensitivity;
consult manual
Proteins may be washed from the
• Reduce the number of washes or reduce the
membrane during assays stringency of washing conditions during
subsequent assay steps
Antigen binding to the membrane • Stain the blot after transfer or use prestained
was insufficient standards to assess transfer efficiency.
Alternatively, use stain-free technology to assess sample binding on the blot. See the previous section for suggestions on improving transfer-related problems
Antigen denaturation occurred during
• Antibodies, especially monoclonals, may not
electrophoresis or transfer recognize denatured antigens
• Electrophorese and transfer proteins under
native conditions. Use a cooling coil and a
refrigerated recirculating bath to transfer
heat-sensitive proteins
Epitope may be blocked by total • Some total protein stains (such as amido
protein stain black and colloidal gold) interfere with antibody
recognition of the antigen. Do not use a total
protein stain or use a different stain or
stain-free technology
The primary or secondary antibody was
• Store the reagents at recommended conditions.
inactive or nonsaturating Avoid repeated freeze-thaw cycles, bacterial
contamination, and heat inactivation
• Detergents may affect the binding of some
antibodies. Eliminate them from the assay,
except for the wash after blocking
• If the antibody titer is too low, optimize the
concentration using a dot-blot experiment
• Increase the antibody incubation times
The enzyme conjugate was inactive • Test the reagent for activity*
or nonsaturating
•Store the reagents at recommended
conditions. Avoid repeated freeze-thaw cycles,
bacterial contamination, and heat inactivation
• Sodium azide is a potent inhibitor of
horseradish peroxidase. Use a different
biocide such as gentamicin sulfate
• Undistilled water may cause inactivation of the
enzyme. Use only distilled, deionized water
• If the conjugate concentration is too low,
optimize using a dot-blot experiment
The color development reagent • Test the reagent for activity* and
was inactive remake if necessary
Regions of poor or uneven signal The membrane was allowed to dry • High intensity or rapid transfer methods
during detection
during handling generate heat. Ensure that warm membranes
are not allowed to dry after transfer
* Tests for Monitoring Reagent Activity
1. Test the activity of the color development solution. Combine 1.0 ml of the color development solution with 10 μl of
full-strength secondary antibody conjugate. The color reaction should occur immediately. If color fails to develop
within a few minutes, the color development solution is inactive. Prepare a fresh working solution and repeat the color
development assay.
2. Test the activity of the conjugate solution. Combine 1.0 ml of the color development solution tested above and 1.0 ml of
the 1:3,000 dilution conjugate solution. A light-blue tinge should develop within 15 min. If color fails to develop within 25
min, the conjugate solution is suspect. Repeat the procedure with a freshly prepared dilution of conjugate.
3. Test the activity of the first antibody solution. Use an ELISA, RID, Ouchterlony immunodiffusion, or precipitation test to
determine reactivity of the antibody with the antigen. If possible, repeat the assay procedure with a more concentrated
primary antibody solution.
67
Protein Blotting Guide
Troubleshooting
Multiscreen Apparatus
ProblemCause
Solution
ProblemCause
Leakage or cross-well contamination
• Tighten the screws using a diagonal
crossing pattern to ensure uniform pressure
on the membrane surface. Do not overtighten
because this will cause the channels to cut
into the membrane
Anionic dyes — low sensitivity
The instrument was assembled incorrectly
The sample template has warped and can no longer provide a proper seal.
(Heating the apparatus to >50°C will
warp the acrylic plates.)
Fluorescent blot stains — low sensitivity
Proteins with low hydrophobicity
Incorrect excitation and emission • Refer to the product literature for correct
settings were used excitation wavelengths and emission filters
• Replace the sample template
Incomplete or uneven filtration
Bubbles trapped within the channels
• Tilt the instrument backward during sample
application to help bubbles rise to the top
Solution
Anionic dye stains do not detect protein • Use a more sensitive stain such as colloidal
bands below ~100 ng gold stain or a fluorescent stain
• Increase the sample load
• Only highly hydrophobic proteins will retain
enough SYPRO stain to be visible on a
membrane.SDS is stripped off proteins during
transfer, resulting in very little retention of the
SYPRO stain on most proteins
• Use slow and careful delivery of reagent to
prevent trapping bubbles inside the channels
Halos around the wells
The membrane was not rehydrated • Rehydrate the membrane prior to loading
after assembly samples. Apply vacuum only until solutions
are removed from the sample wells, then
disconnect the vacuum
Too much protein was loaded, overloading • Determine optimal loading conditions by
the capacity of the membrane performing serial dilutions of samples
The blocking step was too short
• Make sure blocking step is as long as the
longest incubation period
Total Protein Detection
TABLE OF CONTENTS
ProblemCause
Colloidal gold total protein stain — high background
Solution
The blocking step was insufficient • Block with 0.3% Tween 20 in TBS using
or was omitted 3 washes of 20 min each
Contamination occurred during
• Discard and remake the gel and transfer solutions
electrophoresis or transfer
• Replace or thoroughly clean contaminated
fiber pads if a tank blotter was used
Excessive amounts of protein were loaded •Reduce the amount of protein on the gel or
on the gel or too much SDS was used SDS in the transfer buffer
in the transfer buffer. Proteins can pass •Add a second sheet of membrane to bind
through the membrane without binding and excess protein
recirculate through a tank blotting system
The colloidal gold stain solution •Use a separate, clean plastic container to store
was contaminated previously used reagent in the refrigerator
•Discard any reagent that has a viscous
sediment at the bottom of the bottle
•If the solution is no longer dark burgundy but
light blue, discard it. The stain is contaminated
with buffer salts, which react with the gold
solution, causing nonspecific precipitation of
the reagent onto the membrane
The development step was too long
•Overnight development may slightly increase
sensitivity but may also increase background.
Reduce development step to 1–2 hr
Colloidal gold total protein stain — The incubation time was insufficient
• Increase the incubation time for detection of
low sensitivity
low-level signals. Overnight incubation is
possible, although background staining
can increase
68
Transfer was incomplete
• See “Poor electrophoretic transfer” on page 64
The stain was exhausted, as evidenced by the loss of the dark burgundy color
and longer staining times
• Discard the reagent
Buffer salt contamination has occurred; the solution is light blue
instead of dark burgundy
• Discard the reagent
Anionic dyes — high background
Destaining was insufficient
Increase the number and duration of washes
with the destaining solution
Prepare new solution
The dye solution was too concentrated
69
Protein Blotting Guide
Troubleshooting
Appendix
Natural Standards
Recombinant Precision Plus Protein Standards
250
MW,
kD
250
MW,
kD
250
MW,
kD
250
MW,
kD
250
MW,
kD
250
150
150
150
150
150
150
150
100
100
100
100
100
100
100
75
75
75
75
75
75
50
50
50
50
50
37
37
37
37
MW,
kD
250
MW,
kD
250
MW,
kD
150
100
MW,
kD
210
MW,
kD
132
200
75
50
37
25
25
20
Protein Standards for Blotting
n Are useful for monitoring electrophoresis and
transfer efficiency
Protein standards are mixtures of well-characterized
or recombinant proteins that are loaded alongside
protein samples in a gel. Properties and applications
of Bio-Rad’s blotting standards are summarized in
Table A.1 and Figure A.1.
15
Serve as controls to ensure proper location of
transferred bands in repetitive screening experiments
Provide a reference for determining the
molecular weight of proteins identified by
antibody or ligand probes
n
10
TABLE OF CONTENTS
Molecular
Weight Range Molecular
(on Tris-HCI
Weight
Monitoring
or TGX™ gels)
Determination
Electrophoresis
Monitoring
Transfer
Chemiluminescence
Efficiency
Detection
Singleplex
Multiplex
Fluorescence Fluorescence
Detection
Detection
Precision Plus
Prestained Prestained multicolored
Protein™
recombinant fluorescent bands with
WesternC™
integrated Strep-tag for
standards
chemiluminescence
visualization
10–250 kD
•
•
Precision Plus
Prestained
Prestained multicolored
Protein Dual Color recombinant fluorescent bands,
standards
2-color band pattern
10–250 kD
•
•
•
•
Precision Plus
Prestained
Protein Dual Xtra recombinant
standards
2–250 kD
•
•
•
•
10–250 kD
Precision Plus
Protein All Blue
standards
10–250 kD
50
50
66.2
37
37
37
25
25
25
25
20
20
20
20
15
15
15
15
10
5
•
•
•
•
•
•
•
Prestained
Prestained
Prestained
SDS-PAGE
natural
fluorescent bands
standards (natural)
Broad: 7.2–208 kD
High: 47–205 kD
Low: 19–107 kD
Kaleidoscope
Prestained
prestained natural
standards (natural)
7.6–216 kD
•
•
•
•
•
Chemiluminescent Fluorescent
Unstained
Dual Color
Protein standards are available as sets of purified
(natural) or recombinant proteins:
n Natural standards are blended from naturally
occurring proteins to provide a familiar band pattern
on gels and blots
Recombinant standards are engineered with
attributes such as evenly spaced molecular weights
or affinity tags for easy detection; Bio-Rad’s
recombinant standards are available as the Precision
Plus Protein™ standards family.
Unstained standards contain only purified proteins, so
they do not exhibit the variability in molecular weight
sometimes observed with prestained standards.
Therefore, unstained standards or standards with
affinity tags for blot detection deliver high molecular
weight accuracy across a linear fit to a standard
migration curve (r2 >0.99) and are recommended for
the most accurate molecular weight determinations
for gels or blots. Figure A.1 and Table A.2 summarize
the composition and molecular weights of Bio-Rad’s
unstained standards. These standards also image well
after activation on stain-free gels.
Unstained SDS-PAGE Standards
•
45.7
31
35.8
32.5
29
21.5
21
18.4
14.4
7.6
6.9
6.5
Dual Xtra
Kaleidoscope™
All Blue
Unstained
SDS-PAGE
Prestained
SDS-PAGE
Kaleidoscope
Prestained
WesternC™
n
•
56.2
45
10
10
78
101
2
Unstained Standards for Protein Blotting
Precision Plus
Prestained
Prestained
Protein™
recombinantmulticolored
Kaleidoscope™
fluorescent bands,
standards
5-color band pattern
116.3
97.4
10
Prestained standards allow easy and direct
visualization of the separation during electrophoresis
and of their subsequent transfer to membranes
•
70
10
10
n
10–250 kD
Prestained
multicolored fluorescent
bands, multicolored
band pattern
15
Fig. A.1. Bio-Rad’s protein standards for western blotting applications.
Precision Plus
Unstained
Integrated Strep-tag
Protein™ unstained recombinant for chemiluminescence
standards
visualization
Prestained
Prestained
recombinant fluorescent bands
15
n Unstained protein standards offer the most accurate
size determinations
•
Prestained multicolored
fluorescent bands,
2-color band pattern,
extended MW range
15
Prestained
Broad: 6.5–200 kD
High: 45–200 kD
Low: 14.4–97.4 kD
Protein blended
to yield uniform
band intensities
upon staining
25
20
Protein standards are also available either prestained
or unstained:
Table A.1. Protein standards selection guide.
SDS-PAGE
Unstained
unstained natural
standards
(natural)
25
20
125
75
n
Protein standards:
Protein
Standard
Type
Features
10
20
MW,
kD
216
These natural protein standards form tight bands that
transfer reproducibly to membranes. To visualize these
standards, use a total protein stain. If using the blot in
subsequent immunoblotting, use an immunoblottingcompatible stain such as Ponceau S, mark the
positions of the standards on the blot with a pencil,
destain, and then proceed with immunodetection.
SDS-PAGE unstained standards are available in three
molecular weight ranges (Figure A.1 and Table A.2).
Precision Plus Protein Unstained Standards
Precision Plus Protein unstained standards provide a
recombinant ten-band, broad range molecular weight
ladder (10–250 kD). These standards contain an affinity
Strep-tag peptide that displays an intrinsic binding
affinity towards StrepTactin, a genetically modified
form of streptavidin. It is the high-affinity binding of
the Strep-tag sequence to StrepTactin that allows
convenient and simultaneous detection of both proteins
and standards on western blots (Figure A.2) using
either colorimetric or chemiluminescence methods.
Table A.2. Composition and molecular weights (in kD) of Bio-Rad’s unstained standards
for blotting.
Precision Plus Protein Unstained SDS-PAGE
Unstained Standards
Standards
250
150
100
75
50
37
25
20
15
10
High Range Low Range Broad Range
200
—
200
116
—
116.25
97.4
97.4
97.4
66.2
66.2
66.2
45
45
45
—
31
31
—
21.5
21.5
—
14.4
14.4
—
—
6.5
Protein
Myosin
b-galactosidase
Phosphorylase b
BSA
Ovalbumin
Carbonic anhydrase
Trypsin inhibitor
Lysozyme
Aprotinin
71
Protein Blotting Guide
Appendix
Precision Plus Protein™ Prestained Standards
Add
StrepTactin-AP
or -HRP
conjugate
StrepTactin
conjugate binds
Strep-tag sequence
Add substrate
Substrate comes
in contact with
StrepTactin-AP or
-HRP conjugate
TABLE OF CONTENTS
Substrate
conversion
Standard band is
visualized by AP
or HRP enzymatic
release of light
or conversion of
substrate to colored
compound
Precision Plus Protein prestained standards are a blend
of ten recombinant proteins and provide a ten-band,
broad range molecular weight ladder (10–250 kD)
with single (all blue), dual (dual color), or multicolored
(Kaleidoscope™ ) protein bands (Figure A.1); Precision
Plus Protein Dual Xtra protein standards provide an
extended molecular weight range of 2–250 kD (12
bands). The colors allow easy band referencing and
blot orientation.
Because the proteins in the Precision Plus Protein
standards are recombinant and the staining
technology is optimized, molecular weights do not
vary from lot to lot. Dye labeling can be controlled
more effectively, delivering homogeneous staining
and tight, sharp bands. All Precision Plus Protein
prestained standards deliver the most linear fit to
a standard migration curve (r2 >0.99) available for
prestained standards (Figure A.3). As a result these
standards may be used for highly accurate estimation
of molecular weight across a broad size range.
Kaleidoscope Standards
Kaleidoscope prestained standards contain individually
colored proteins that allow instant band recognition
on western blots or gels. The proteins are labeled with
fluorescent dyes and so can be used in fluorescence
detection applications.
Fig. A.2. Overview of the StrepTactin detection system.
Prestained SDS-PAGE Standards
Prestained Standards for Western Blotting
Naturally occurring prestained SDS-PAGE standards
are available in specific size ranges: low, high, and
broad (Table A.2).
The ability to visualize prestained standards during
electrophoresis makes them ideal for monitoring protein
separation during PAGE. The ease in transferring
to the blot also make them popular for monitoring
transfer efficiency and the general location of antigens
in repetitive screening assays (Tsang et al. 1984).
This, combined with recent improvements made in
their design and manufacture, has made prestained
standards an excellent choice for estimations of
molecular weights on western blots. Bio-Rad provides
both recombinant and natural prestained standards
(Figure A.1 and Table A.2).
2.5
2.0
log MW
Individual Precision
Plus Protein
standard with
integrated Strep-tag
sequence
1.5
1.0
0.5
0.0
0.20.4 0.6 0.8 1.0
Rf
Fig. A.3. Exceptional linearity of Precision Plus Protein™
standards. The standard curve was generated by plotting the log
molecular weight (MW) vs. the relative migration distance (R f) of
each protein standard band through an SDS-PAGE gel. Precision
Plus Protein™ Kaleidoscope™ standards showed r 2 = 0.996,
demonstrating a very linear standard curve.
Precision Plus Protein™ WesternC™ Standards
Precision Plus Protein WesternC standards were
designed for western blotting applications. Like the rest
of the Precision Plus Protein family of standards, the
WesternC standards contain ten bands of 10–250 kD
(Figure A.1). Unique to WesternC standards is the
combination of both unstained and prestained bands
that migrate in identical fashion. Having both unstained
and prestained bands enables:
n
n
n
Monitoring of the progression of
gel electrophoresis
Monitoring transfer efficiency
Molecular weight determination
(after blot development)
These standards have a Strep-tag affinity peptide to
enable chemiluminescence detection when probed
with StrepTactin-HRP conjugates (Figure A.2), so
the protein standard appears directly on a film or
CCD image. In addition, the prestained bands have
fluorescence properties and so can be used in
fluorescence detection applications.
Links
Unstained SDS-PAGE
Standards
Precision Plus Protein
Unstained Standards
Prestained SDS-PAGE
Standards
Precision Plus Protein
Prestained Standards
Precision Plus Protein
WesternC Standards
72
73
Protein Blotting Guide
Troubleshooting
Glossary
Colorimetric detection Detection of molecules of interest by formation of a colored product
Conjugate Enzyme-antibody compound used in blotting
Coomassie Blue
Anionic dye used in the total protein staining of gels and blots
Diaminobenzidine (DAB) Color development reagent used with HRP and other peroxidases that produces an
insoluble brown reaction product at the site of the peroxidase-antibody complex
Dot blot Direct application of proteins in free solution to a membrane
Dunn buffer Commonly used transfer buffer (10 mM NaHCO3, 3 mM Na2CO3, 20% methanol,
pH 9.9)
4-Chloro-1-naphthol (4CN) Color development reagent used with horseradish peroxidase (HRP), which produces
an insoluble purple reaction product at the site of an enzyme-antibody complex
5-Bromo-4-chloro-indolyl Color development reagent used with alkaline phosphatase (AP), which in the
phosphate (BCIP) presence of NBT produces an insoluble purple reaction product at the site of the
enzyme-antibody complex
Alkaline phosphatase (AP) Enzyme used as a detection reagent, usually conjugated to a secondary
antibody probe
TABLE OF CONTENTS
Amido black 10B Anionic dye used in the total protein detection of blots
Amplified AP kit Highly sensitive detection kit that utilizes a streptavidin-biotin system
Anionic dye Negatively charged compound used as a stain; used in blotting to stain proteins
immobilized on nitrocellulose or PVDF membranes
Use of the driving force of an electric field to move proteins from gels to membranes
Enzyme conjugate Enzyme covalently attached to another protein; in blotting, usually an antibody
Foam pad Pad used in blotter cassettes that helps hold the gel and membrane sandwich
in place
Filter paper Cotton fiber paper used in blotting applications and gel drying
Gelatin Protein commonly used as a blocking reagent in western blotting procedures
High-intensity transfer High-power blotting option. These transfers speed up the blotting process but
produce heat and may cause proteins to migrate through the membrane
Horseradish peroxidase (HRP) Enzyme used in the specific detection of molecules on blots, usually conjugated to a
secondary antibody probe
Immunoassay Test for a substance by its reactivity with an antibody
Immunoblotting Blot detection by antibody binding
Immunodetection Detection of a molecule by its binding to an antibody
Antibody Immunoglobulin (IgG); protein produced in response to an antigen that specifically
binds the portion of the antigen that initiated its production
Immunoglobulin (IgG)Antibody; protein produced in response to an antigen that specifically binds the
portion of the antigen that initiated its production
Antigen Molecule that specifically binds with an antibody
Ligand Assay Analysis of the quantity or characteristics of a substance
Avidin Glycoprotein found in egg white that binds biotin with high specificity
Membrane Immobilizing support medium used in blotting, generally in the form of a sheet that
has high affinity for biological molecules; for example, nitrocellulose or PVDF
Background Nonspecific signal or noise that can interfere with the interpretation of valid signals
Biotin Small molecule that binds specifically to avidin or streptavidin
Bjerrum
Schafer-Nielsen buffer
Commonly used transfer buffer (48 mM Tris, 39 mM glycine, 20% methanol, pH 9.2)
Blocking reagent Protein used to saturate unoccupied binding sites on a blot to prevent nonspecific
binding of antibody or protein probes to the membrane
BlotImmobilization of proteins or other molecules onto a membrane; or, the membrane
that has the molecules adsorbed onto its surface
BLOTTO Formulation of nonfat milk used to block nonspecific binding of proteins
to membranes
Chemiluminescence Emission of light due to a chemical reaction; used in the specific detection of
blotted molecules
Colloidal gold Stabilized solution of gold particles; used as a blot detection reagent when
conjugated to antibodies or ligands. It produces a rose-red color on the membrane
at the site of deposition
Color development reagent
74
Electrophoretic blotting Enzyme substrate used in blotting to visualize the location of an
enzyme-antibody complex
Membrane/filter paper sandwiches
Molecule that binds another in a complex
Blotting membrane and filter paper precut for a specific gel size
Microfiltration blotting Use of a microfiltration device, such as the Bio-Dot® apparatus, to immobilize protein
in free solution onto a membrane
Multiplexing Blotting technique that allows identification of two or more bands on a membrane
without having to strip and reprobe
Multiscreen apparatus Instrument that allows the screening of two blots with up to 40 different
antibody samples
Native PAGE Version of PAGE that retains native protein configuration, performed in absence of
SDS and other denaturing agents
NHS-biotin N-hydroxysuccinimide-biotin, a reagent that biotinylates proteins
Nitroblue tetrazolium Color development reagent used with AP, which with BCIP produces an insoluble
(NBT) purple reaction product at the site of the AP-antibody complex
Nitrocellulose General-purpose blotting membrane
Nonenzymatic probe Molecule used in blot detection that does not involve an enzyme-catalyzed reaction;
for example, a radioactive, chemiluminescent, or colloidal gold-labeled molecule
75
Protein Blotting Guide
Troubleshooting
Glossary
Nonfat dry milk Material used in solution as a blocking reagent for western blots
Nonspecific binding Interaction between bound proteins and probes that is not a result of a specific
reaction; results in spurious signals on the membrane
PAGE Polyacrylamide gel electrophoresis, a common method of separating proteins
Phycobiliprotein Protein from the light-harvesting complex of some algae. The fluorescent properties
of these proteins make ideal fluorescence detection agents for blotting, when
coupled to immunoglobulin
Polyvinylidene difluoride (PVDF) Membrane used in protein blotting that has high chemical resistance, tensile strength,
binding, and retentive capacity, making it ideal for use in protein sequencing
Power supply Instrument that provides the electric power to drive electrophoresis and
electrophoretic blotting experiments
Primary antibody Antibody that binds a molecule of interest
Prestained standards Mixture of molecular weight marker proteins that have covalently attached dye
molecules, which render the bands visible during electrophoresis and transfer; used
to assess the transfer efficiency of proteins onto a membrane
Probe Supported nitrocellulose High tensile–strength blotting membrane; nitrocellulose that has been cast on an
inert high-strength support
Tank blotting Use of a tank blotting apparatus, which consists of a tank of buffer with vertically
oriented platinum wire or plate electrodes; the gel and membrane are held in place
between the electrodes by a porous cassette
Total protein stain Reagent that binds nonspecifically to proteins; used to detect the entire protein
pattern on a blot or gel
Towbin buffer Common protein blotting transfer buffer (25 mM Tris, pH 8.5, 192 mM glycine,
20% methanol)
Transfer Immobilization of proteins or other molecules onto a membrane by electrophoretic
or passive means
Tween 20 Nonionic detergent; used in blot detection procedures as a blocking reagent or
added to wash buffers to minimize nonspecific binding and background
Western blotting Immobilization of proteins onto a membrane and subsequent detection by
protein-specific binding and detection reagents
A molecule used to specifically identify another molecule
Protein A Protein derived from Staphylococcus aureus that binds a wide range of
immunoglobulins from various species
TABLE OF CONTENTS
Protein G Protein derived from Streptococcus that binds a wide range of immunoglobulins from
various species and has a wider range of binding capabilities than protein A
Rapid semi-dry blotting Semi-dry blotting technique that uses increased current density to transfer
biomolecules more efficiently than other techniques
SDS-PAGE Separation of molecules by molecular weight in a polyacrylamide gel matrix in the
presence of a denaturing detergent, sodium dodecyl sulfate (SDS)
Secondary antibody Antibody that binds a primary antibody; used to facilitate detection
Semi-dry blotting Use of a semi-dry blotting apparatus, which consists of two horizontally oriented
plate electrodes. The gel and membrane sandwich is positioned between the
electrodes with buffer-soaked filter paper on either side of the sandwich which
serve as buffer reservoirs
Signal-to-noise ratio Relative difference in detection level between the specific and background signals
Stain-free technologyProtein detection technology involving UV-induced haloalkane modification of protein
tryptophan residues. Continued exposure to UV light causes fluorescence of the
modified proteins, which are then detected by a CCD imager. Sensitivity of this
technique is generally equal to or better than Coomassie staining
StrepTactin Genetically engineered form of streptavidin, used with the Precision Plus Protein™
Unstained standards for detection
Strep-tag sequence Amino acid sequence that can be used to tag a protein, enabling its detection by
StrepTactin binding; this sequence is present in Precision Plus Protein Unstained
and WesternC™ standards
Streptavidin Protein that binds biotin with high affinity; generally regarded as superior to avidin
because it is not glycosylated
Substrate Substance that is reacted upon by an enzyme; for example, a color
development reagent
Super cooling coil Optional accessory of the Trans-Blot® cell that can be attached to a refrigerated
water recirculator to cool the buffer during high-intensity transfers
76
77
References
Protein Blotting Guide
polyacrylamide gel. Anal Biochem 111, 385–392.
Moeremans M et al. (1987). The use of colloidal metal particles in protein blotting. Electrophoresis 8, 403–409.
Reinhart MP and Malamud D (1982). Protein transfer from isoelectric focusing gels: the native blot. Anal Biochem 123, 229–235.
Rohringer R and Holden DW (1985). Protein blotting: detection of proteins with colloidal gold, and of glycoproteins and lectins with
biotin-conjugated and enzyme probes. Anal Biochem 144, 118–127.
Tovey ER and Baldo BA (1987). Comparison of semi-dry and conventional tank-buffer electrotransfer of proteins from polyacrylamide gels
to nitrocellulose membranes. Electrophoresis 8, 384–387.
Tovey ER and Baldo BA (1987). Characterisation of allergens by protein blotting. Electrophoresis 8, 452–463.
Towbin H et al. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc Natl Acad Sci USA 76, 4350–4354.
Tsang VC et al. (1984). Calibration of prestained protein molecular weight standards for use in the “Western” or enzyme-linked
immunoelectrotransfer blot techniques. Anal Biochem 143, 304–307.
References
Akerstrom B et al. (1985). Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies.
J Immunol 135, 2589–2592.
Bayer EA and Wilchek M (1980). The use of the avidin-biotin complex as a tool in molecular biology. Methods Biochem Anal 26, 1–45.
Tsang VC et al. (1985). Enzyme-linked immunoelectrotransfer blot (EITB). In Enzyme-Mediated Immunoassay. T.T. Ngo and H.M. Lenhoff, eds.
(New York: Plenum Press), 389–414.
Turner BM (1983). The use of alkaline-phosphatase-conjugated second antibody for the visualization of electrophoretically separated proteins
recognized by monoclonal antibodies. J Immunol Methods 63, 1–6.
Wisdom GB (1994). Protein blotting. Methods Mol Biol 32, 207–213.
Beisiegel U (1986). Protein blotting. Electrophoresis 7, 1–18.
TABLE OF CONTENTS
Bers G and Garfin D (1985). Protein and nucleic acid blotting and immunobiochemical detection. Biotechniques 3, 276–288.
Related Reading
Bjerrum OJ and Schafer-Nielsen C (1986). Buffer systems and transfer parameters for semidry electroblotting with a horizontal apparatus.
In Electrophoresis ’86: Proceedings of the Fifth Meeting of the International Electrophoresis Society, M.J. Dunn, ed. (Weinheim, Germany:
Wiley-VCH Verlag GmbH), 315–327.
Protein Electrophoresis: A guide to polyacrylamide gel electrophoresis (PAGE) and detection. Bio-Rad Bulletin 6040.
Blake MS et al. (1984). A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots.
Anal Biochem 136, 175–179.
Sansan L et al. (2010). Precision Plus Protein Dual Xtra standards — new protein standards with an extended range from 2 to 250 kD.
Bio-Rad Bulletin 5956.
Boyle MDP and Reis KJ (1987). Bacterial Fc receptors. Biotechnology 5, 697–703.
Western Blotting Troubleshooter. Bio-Rad Bulletin 1529.
Burnette WN (1981). “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified
nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112, 195–203.
Chimento DP et al. (2009). Enhanced multiplex fluorescent western blotting. Bio-Rad Bulletin 5881.
Carr DW and Scott JD (1992). Blotting and band-shifting: techniques for studying protein-protein interactions.
Trends Biochem Sci 17, 246–249.
Elbaggari A et al. (2008). Evaluation of the Criterion Stain Free gel imaging system for use in western blotting applications.
Bio-Rad Bulletin 5781.
Chaiet L and Wolf FJ (1964). The properties of streptavidin, a biotin-binding protein produced by Streptomycetes.
Arch Biochem Biophys 106, 1–5.
Elbaggari A et al. (2008). Imaging of chemiluminescent western blots: comparison of digital imaging and X-ray film. Bio-Rad Bulletin 5809.
Crisp SJ and Dunn MJ (1994). Detection of proteins on protein blots using chemiluminescent systems. Methods Mol Biol 32, 233–237.
Dunn MJ (1994). Detection of proteins on blots using the avidin-biotin system. Methods Mol Biol 32, 227–232.
Dunn MJ (1999). Detection of total proteins on western blots of 2-D polyacrylamide gels. Methods Mol Biol 112, 319–329.
Egger D and Bienz K (1994). Protein (western) blotting. Mol Biotechnol 1, 289–305.
Garfin DE and Bers G (1989). Basic aspects of protein blotting. In Protein Blotting: Methodology, Research and Diagnostic Applications,
B.A. Baldo et al., eds. (Basel, Switzerland: Karger), 5–42.
Gershoni JM (1985). Protein blotting: developments and perspectives. Trends Biochem Sci 10, 103–106.
Tan A (1999). Increased transfer efficiency using a discontinuous buffer system with the Trans-Blot SD semi-dry electrophoretic transfer cell.
Bio-Rad Bulletin 2134.
McDonald K et al. (2005). Fluorescent nanoparticles for western blotting. Bio-Rad Bulletin 3179.
Taylor S et al. (2008). The dynamic range effect of protein quantitation in polyacrylamide gels and on western blots. Bio-Rad Bulletin 5792.
Increase western blot throughput with multiplex fluorescent detection. (2010). Bio-Rad Bulletin 5723.
Strep-tag technology for molecular weight (MW) determinations on blots using Precision Plus Protein™ standards. (2010). Bio-Rad Bulletin 2847.
Urban M and Woo L (2010). Molecular weight estimation and quantitation of protein samples using Precision Plus Protein WesternC standards,
the Immun-Star WesternC chemiluminescent detection kit, and the ChemiDoc XRS imaging system.
Bio-Rad Bulletin 5576.
Lai L et al. (2010). Molecular weight estimation using Precision Plus Protein WesternC standards on Criterion Tris-HCl and Criterion XT Bis-Tris
gels. Bio-Rad Bulletin 5763.
Gershoni JM (1987). Protein blotting: a tool for the analytical biochemist. In Advances in Electrophoresis, Vol 1, A. Chrambach et al., eds.
(Weinheim, Germany: Wiley-VCH Verlag GmbH), 141–175.
Gershoni JM (1988). Protein blotting: a manual. Methods Biochem Anal 33, 1–58.
Gershoni JM and Palade GE (1983). Protein blotting: principles and applications. Anal Biochem 131, 1–15.
Gershoni JM et al. (1985). Protein blotting in uniform or gradient electric fields. Anal Biochem 144, 32–40.
Goding JW (1978). Use of staphylococcal protein A as an immunological reagent. J Immunol Methods 20, 241–253.
Gooderham K (1984). Transfer techniques in protein blotting. Methods Mol Biol 1, 165–178.
Guesdon J-L et al. (1979). The use of avidin-biotin interaction in immunoenzymatic techniques. J Histochem Cytochem 27, 1131–1139.
Harper DR et al. (1990). Protein blotting: ten years on. J Virol Methods 30, 25–39.
Hawkes R et al. (1982). A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem 119, 142–147.
Hsu SM et al. (1981). Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and
unlabeled antibody (PAP) procedures. J Histochem Cytochem 29, 577–580.
Kurien BT and Scofield RH (2003). Protein blotting: a review. J Immunol Methods 274, 1–15.
Kyhse-Andersen J (1984). Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from
polyacrylamide to nitrocellulose. J Biochem Biophys Methods 10, 203–209.
Langone JJ (1982). Use of labeled protein A in quantitative immunochemical analysis of antigens and antibodies. J Immunol Methods 51, 3–22.
Legocki and Verma (1981). Multiple immunoreplica technique: screening for specific proteins with a series of different antibodies using one
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Ordering Info
Protein Blotting Guide
Ordering
Information
170-4071 Criterion Blotter with Wire Electrodes, includes
buffer tank assembled with wire electrodes, lid
with cables, 2 gel holder cassettes, 4 foam pads,
1 pack precut blot absorbent filter paper, gel/blot
assembly tray, roller, sealed ice block
165-6024 Criterion Cell/Plate Blotter System,
includes Criterion cell and Criterion blotter
with plate electrodes
165-6025 Criterion Cell/Wire Blotter System,
includes Criterion cell and Criterion blotter
with wire electrodes
170-3872 Criterion Blotter with Plate Electrodes and
PowerPac HC Power Supply
170-3874 Criterion Blotter with Wire Electrodes and
PowerPac HC Power Supply
Ordering Information
Electrophoretic Transfer Cells
Catalog # Description
TABLE OF CONTENTS
Trans-Blot® Cells and Systems
170-3939 Trans-Blot Cell with Plate Electrodes and Super
Cooling Coil, includes 2 gel holder cassettes,
buffer tank, lid with power cables, 4 foam pads,
1 pack precut blot absorbent filter paper
(15 x 20 cm)
170-3853 Trans-Blot Cell with Plate Electrodes, Super
Cooling Coil, and PowerPac™ HC Power Supply
(100–120/220–240V)
170-3946 Trans-Blot Cell with Plate Electrodes, includes
2 gel holder cassettes, buffer tank, lid with
power cables, 4 foam pads, 1 pack precut blot
absorbent filter paper (15 x 20 cm)
170-3850 Trans-Blot Cell with Plate Electrodes and
PowerPac HC Power Supply (100–120/220–240V)
170-3910 Trans-Blot Cell with Wire Electrodes, includes
2 gel holder cassettes, buffer tank, lid with
power cables, 4 foam pads, 1 pack precut blot
absorbent filter paper (15 x 20 cm)
170-3825 Trans-Blot Cell with Wire Electrodes and PowerPac
HC Power Supply (100–120/220–240V)
Trans-Blot Cell Accessories
170-3914 Foam Pads, 15.5 x 20.5 cm, 6 pads
170-3956 Thick Blot Paper, 15 x 20 cm, for Trans-Blot
cassette, 25 sheets
170-3960 Extra Thick Blot Paper, 15 x 20 cm, 30 sheets
170-3943 Trans-Blot Platinum Anode Plate Electrode
170-3944 Trans-Blot Stainless-Steel Cathode Plate Electrode
170-3945 Trans-Blot Plate Electrode Pair, platinum anode
and stainless-steel cathode
170-3920 Trans-Blot Standard Wire Electrode Card, cathode
170-3921 Trans-Blot Standard Wire Electrode Card, anode
170-3912 Super Cooling Coil, 1 required for all
high-intensity transfers
170-3913 Gel Holder Cassette, includes 2 fiber pads
170-3922 Trans-Blot Cell Buffer Tank
170-3923 Trans-Blot Cell Lid with Power Cables
Trans-Blot Plus Cell and Systems
170-3990 Trans-Blot Plus Cell with Plate Electrodes
and Super Cooling Coil, includes 3 gel holder
cassettes, buffer tank, lid with power cables, 6
foam pads, 1 pack blot absorbent filter paper
(26.5 x 28 cm, 30 sheets), roller, stirbar
170-3991 Trans-Blot Plus Cell with Plate Electrodes, Super
Cooling Coil, and PowerPac HC Power Supply
(100–120/220–240V)
170-3992 Trans-Blot Plus Cell with Plate Electrodes, Super
Cooling Coil, and PowerPac Universal Power Supply
(100–120/220–240V)
80
Trans-Blot Plus Cell Accessories
170-3994 Trans-Blot Plus Gel/Cassette Assembly Tray
170-3995 Foam Pads, 27 x 28.5 cm, 2 pads
170-3997 Stirbar
170-3998 Trans-Blot Plus Roller, 6 in. wide
170-3999 Trans-Blot Plus Gel Holder Cassette with Clamps
170-4990 Trans-Blot Plus Super Cooling Coil
170-4991
Trans-Blot Plus Platinum Anode Plate Electrode
170-4992 Trans-Blot Plus Stainless-Steel Cathode
Plate Electrode
170-4995 Trans-Blot Plus Cell Buffer Tank
170-4996 Trans-Blot Plus Cell Lid with Cables
170-4997 Gel Holder Cassette Clamps, for Trans-Blot Plus cell,
3 clamps
Mini Trans-Blot® Cell and Systems
170-3930 Mini Trans-Blot Electrophoretic Transfer Cell,
includes 2 gel holder cassettes, 4 foam pads,
modular electrode assembly, blue cooling unit,
lower buffer tank, lid with cables
170-3935 Mini Trans-Blot Module, without lower buffer
tank and lid
170-3989 Mini Trans-Blot Cell and PowerPac Basic Power
Supply (100–120/220–240V)
170-3836 Mini Trans-Blot Cell and PowerPac HC Power
Supply (100–120/220–240V)
165-8029 Mini-PROTEAN® Tetra Cell and Mini Trans-Blot
Module, includes 10-well, 1.0 mm, 4-gel system
and blotting module without lower buffer tank and
lid; gel casting accessories
165-8033 Mini-PROTEAN Tetra Cell, Mini Trans-Blot
Module, and PowerPac Basic Power Supply
165-8034 Mini-PROTEAN Tetra Cell for Mini Precast Gels,
Mini Trans-Blot Module, and PowerPac Basic
Power Supply
165-8036 Mini-PROTEAN Tetra Cell for Mini Precast Gels,
Mini Trans-Blot Module, and PowerPac HC
Power Supply
165-8035 Mini-PROTEAN Tetra Cell, Mini Trans-Blot Module,
and PowerPac HC Power Supply
Mini Trans-Blot Cell Accessories
170-3931 Mini Gel Holder Cassette
170-3932 Thick Blot Paper, 7.5 x 10 cm, for Mini Trans-Blot
cassette, 50 sheets
170-3933 Foam Pads, 8 x 11 cm, 4 pads
170-3812 Mini Trans-Blot Central Core
170-3919 Blue Cooling Unit, for Mini-PROTEAN Tetra tanks
170-3934 Bio-Ice™ Cooling Unit, for Mini-PROTEAN 3 tanks
Criterion™ Blotters and Systems
170-4070 Criterion Blotter with Plate Electrodes, includes
buffer tank assembled with plate electrodes, lid
with cables, 2 gel holder cassettes, 4 foam pads,
1 pack precut blot absorbent filter paper, gel/blot
assembly tray, roller, sealed ice block
Criterion Blotter Accessories
170-4076 Optional Criterion Blotter Cooling Coil
170-4080 Criterion Blotter Gel Holder Cassette
170-4081 Criterion Blotter Platinum Anode Plate Electrode
170-4082 Criterion Blotter Stainless-Steel Cathode
Plate Electrode
170-4083 Criterion Blotter Wire Electrode Card, anode
170-4084 Criterion Blotter Wire Electrode Card, cathode
170-4085 Thick Blot Paper, 9.5 x 15.2 cm, 50 sheets
170-4086 Criterion Blotter Foam Pad, 9.5 x 15.2 cm, 4 pads
170-4087 Sealed Ice Block, 2 blocks
170-4089 Criterion Gel/Blot Assembly Tray
165-1279 Roller
170-4077 Criterion Blotter Buffer Tank
170-4079 Criterion Blotter Lid with Cables
Trans-Blot SD Semi-Dry Cell and Systems
170-3940 Trans-Blot SD Semi-Dry Electrophoretic Transfer
Cell, includes Trans-Blot SD transfer cell,
Trans-Blot SD agarose gel support frame,
extra thick blot paper
170-3848 Trans-Blot SD Cell and PowerPac HC Power Supply
170-3849 Trans-Blot SD Cell and PowerPac Universal
Power Supply
Trans-Blot SD Cell Accessories
170-3947 Cathode Plate, stainless-steel upper electrode
170-3942 Anode Plate, platinum-coated lower electrode
170-3966 Extra Thick Blot Paper, for Mini-PROTEAN 3 or
Ready Gel® precast gels, 7 x 8.4 cm, 60 sheets
170-3967 Extra Thick Blot Paper, for Criterion gels, 8 x 13.5
cm, 60 sheets
170-3968 Extra Thick Blot Paper, for PROTEAN® II xi gels,
14 x 16 cm, 30 sheets
170-3969 Extra Thick Blot Paper, for PROTEAN II XL gels,
19 x 18.5 cm, 30 sheets
Trans-Blot® Turbo™ Blotting System
170-4155 Trans-Blot Turbo Starter System, includes
Turbo system and starter kit
Trans-Blot Turbo Accessories
170-4156Trans-Blot Turbo Mini PVDF Transfer Packs, pkg of 10, 7 x 8.5 cm, precut blotting transfer pack,
includes filter paper, buffer, PVDF membrane
170-4157Trans-Blot Turbo Midi PVDF Transfer Packs, pkg
of 10, 8.5 x 13.5 cm, precut blotting transfer pack,
includes filter paper, buffer, PVDF membrane
170-4158Trans-Blot Turbo Mini Nitrocellulose Transfer
Packs, pkg of 10, 7 x 8.5 cm, precut blotting
transfer pack, includes filter paper, buffer,
nitrocellulose membrane
170-4159Trans-Blot Turbo Midi Nitrocellulose Transfer
Packs, pkg of 10, 8.5 x 13.5 cm, precut blotting
transfer pack, includes filter paper, buffer,
nitrocellulose membrane
170-4152
Trans-Blot Turbo Base
170-4151
Trans-Blot Turbo Cassette
Microfiltration Apparatus
Bio-Dot® Apparatus and Systems
170-3938 Bio-Dot Microfiltration System, includes
Bio-Dot apparatus and Bio-Dot SF module
templates, vacuum manifold base, gasket
support plates, gasket
170-6545 Bio-Dot Apparatus, includes Bio-Dot sample
template, vacuum manifold base, gasket support
plate, gasket
170-6547 Bio-Dot Module, without vacuum manifold base;
for conversion of Bio-Dot SF to Bio-Dot apparatus
170-6542 Bio-Dot SF Apparatus, includes Bio-Dot SF
sample template, vacuum manifold base, gasket
support plate, gasket, filter paper
170-6543 Bio-Dot SF Module, without vacuum manifold base,
for conversion of Bio-Dot to Bio-Dot SF apparatus
Bio-Dot System Accessories
170-6546 Bio-Dot Gaskets, 3 gaskets
170-6544 Bio-Dot SF Gaskets, 2 gaskets
162-0161 Bio-Dot/Bio-Dot SF Filter Paper, 11.3 x 7.7 cm,
60 sheets
Power Supplies
164-5050 PowerPac Basic Power Supply (100–120/220–240V)
164-5052 PowerPac HC Power Supply (100–120/220–240V)
164-5070 PowerPac Universal Power Supply
Membranes
Nitrocellulose Membrane (0.45 μm)
162-0115 Nitrocellulose Membrane, 0.45 µm,
30 cm x 3.5 m, 1 roll
162-0251 Nitrocellulose Membranes, 0.45 µm,
26.5 x 28 cm, 10 sheets
162-0113 Nitrocellulose Membranes, 0.45 µm,
20 x 20 cm, 5 sheets
162-0116Nitrocellulose Membranes, 0.45 µm,
15 x 15 cm, 10 sheets
162-0114Nitrocellulose Membranes, 0.45 µm,
15 x 9.2 cm, 10 sheets
162-0148 Nitrocellulose Membranes, 0.45 µm,
11.5 x 16 cm, 10 sheets
162-0117 Nitrocellulose Membranes, 0.45 µm,
9 x 12 cm, 10 sheets
162-0167 Nitrocellulose Membranes, 0.45 µm,
8.5 x 13.5 cm, 10 sheets
162-0145 Nitrocellulose Membranes, 0.45 µm,
7 x 8.4 cm, 10 sheets
162-0234 Nitrocellulose/Filter Paper Sandwiches, 0.45 µm,
8.5 x 13.5 cm, 20 pack
162-0235 Nitrocellulose/Filter Paper Sandwiches, 0.45 µm,
8.5 x 13.5 cm, 50 pack
162-0214 Nitrocellulose/Filter Paper Sandwiches, 0.45 µm,
7 x 8.5 cm, 20 pack
162-0215 Nitrocellulose/Filter Paper Sandwiches, 0.45 µm,
7 x 8.5 cm, 50 pack
Nitrocellulose Membrane (0.2 μm)
162-0112 Nitrocellulose Membrane, 0.2 µm,
30 cm x 3.5 m, 1 roll
162-0252 Nitrocellulose Membranes, 0.2 µm,
26.5 x 28 cm, 10 sheets
162-0150 Nitrocellulose Membranes, 0.2 µm,
20 x 20 cm, 5 sheets
162-0147 Nitrocellulose Membranes, 0.2 µm,
13.5 x 16.5 cm, 10 sheets
162-0168 Nitrocellulose Membranes, 0.2 µm,
8.5 x 13.5 cm, 10 sheets
162-0146 Nitrocellulose Membranes, 0.2 µm,
7 x 8.4 cm, 10 sheets
162-0232 Nitrocellulose/Filter Paper Sandwiches,
0.2 µm, 8.5 x 13.5 cm, 20 pack
162-0233 Nitrocellulose/Filter Paper Sandwiches,
0.2 µm, 8.5 x 13.5 cm, 50 pack
81
Ordering Info
Protein Blotting Guide
162-0212 Nitrocellulose/Filter Paper Sandwiches, 0.2 µm,
7 x 8.5 cm, 20 pack
162-0213 Nitrocellulose/Filter Paper Sandwiches, 0.2 µm,
7 x 8.5 cm, 50 pack
162-0216 Sequi-Blot PVDF/Filter Paper Sandwiches,
7 x 8.5 cm, 20 pack
162-0217 Sequi-Blot PVDF/Filter Paper Sandwiches,
7 x 8.5 cm, 50 pack
Supported Nitrocellulose Membrane (0.45 μm)
162-0094 Supported Nitrocellulose Membrane, 0.45 µm,
30 cm x 3 m, 1 roll
162-0254 Supported Nitrocellulose Membranes, 0.45 µm,
26.5 x 28 cm, 10 sheets
162-0093 Supported Nitrocellulose Membranes, 0.45 µm,
20 x 20 cm, 10 sheets
Blotting Membrane/Filter Paper Sandwiches
Mini-PROTEAN Membrane/Filter Paper Sandwiches
162-0212 0.2 μm Nitrocellulose/Filter Paper Sandwiches,
7 x 8.5 cm, 20 pack
162-0213 0.2 μm Nitrocellulose/Filter Paper Sandwiches,
7 x 8.5 cm, 50 pack
162-0214 0.45 μm Nitrocellulose/Filter Paper Sandwiches,
7 x 8.5 cm, 20 pack
162-0092 Supported Nitrocellulose Membranes, 0.45 µm,
15 x 15 cm, 10 sheets
162-0091 Supported Nitrocellulose Membranes, 0.45 µm,
10 x 15 cm, 10 sheets
162-0070 Supported Nitrocellulose Membranes, 0.45 µm,
8.5 x 13.5 cm, 10 sheets
162-0090 Supported Nitrocellulose Membranes, 0.45 µm,
7 x 8.4 cm, 10 sheets
TABLE OF CONTENTS
Supported Nitrocellulose Membrane (0.2 μm)
162-0097 Supported Nitrocellulose Membrane, 0.2 µm,
30 cm x 3 m, 1 roll
162-0253 Supported Nitrocellulose Membranes, 0.2 µm,
26.5 x 28 cm, 10 sheets
162-0096 Supported Nitrocellulose Membranes, 0.2 µm,
15 x 15 cm, 10 sheets
162-0071 Supported Nitrocellulose Membranes, 0.2 µm,
8.5 x 13.5 cm, 10 sheets
162-0095 Supported Nitrocellulose Membranes, 0.2 µm,
7 x 8.4 cm, 10 sheets
Immun-Blot® LF PVDF Membrane
162-0264 Immun-Blot LF PVDF Membrane, pkg of 1 roll,
0.2 μm, 26.5 cm x 3.75 m
162-0260 Immun-Blot LF PVDF/Filter Paper, pkg of 10,
7 x 8.5 cm
162-0261 Immun-Blot LF PVDF/Filter Paper, pkg of 20,
7 x 8.5 cm
162-0262 Immun-Blot LF PVDF/Filter Paper, pkg of 10
8.5 x 13.5 cm
162-0263 Immun-Blot LF PVDF/Filter Paper, pkg of 20,
8.5 x 13.5 cm
Immun-Blot PVDF Membrane
162-0177 Immun-Blot PVDF Membrane, 26 cm x 3.3 m, 1 roll
162-0255 Immun-Blot PVDF Membrane, 25 x 28 cm,
10 sheets
162-0176 Immun-Blot PVDF Membranes, 20 x 20 cm,
10 sheets
162-0175Immun-Blot PVDF Membranes, 10 x 15 cm,
10 sheets
162-0174 Immun-Blot PVDF Membranes, 7 x 8.4 cm,
10 sheets
162-0238 Immun-Blot PVDF/Filter Paper Sandwiches,
8.5 x 13.5 cm, 20 pack
162-0239 Immun-Blot PVDF/Filter Paper Sandwiches,
8.5 x 13.5 cm, 50 pack
162-0218 Immun-Blot PVDF/Filter Paper Sandwiches,
7 x 8.5 cm, 20 pack
162-0219 Immun-Blot PVDF/Filter Paper Sandwiches,
7 x 8.5 cm, 50 pack
Sequi-Blot™ PVDF Membrane
162-0184 Sequi-Blot PVDF Membrane, 26 cm x 3.3 m, 1 roll
162-0256 Sequi-Blot PVDF Membranes, 25 x 28 cm, 10 sheets
162-0182 Sequi-Blot PVDF Membranes, 20 x 20 cm, 10 sheets
162-0181 Sequi-Blot PVDF Membranes, 15 x 15 cm, 10 sheets
162-0180 Sequi-Blot PVDF Membranes, 10 x 15 cm, 10 sheets
162-0186 Sequi-Blot PVDF Membranes, 7 x 8.4 cm, 10 sheets
162-0236 Sequi-Blot PVDF/Filter Paper Sandwiches,
8.5 x 13.5 cm, 20 pack
162-0237 Sequi-Blot PVDF/Filter Paper Sandwiches,
8.5 x 13.5 cm, 50 pack
82
162-0215 0.45 μm Nitrocellulose/Filter Paper Sandwiches,
7 x 8.5 cm, 50 pack
162-0260 Immun-Blot LF PVDF/Filter Paper,
7 x 8.5 cm, 10 pack
162-0261 Immun-Blot LF PVDF/Filter Paper,
7 x 8.5 cm, 20 pack
162-0218 Immun-Blot PVDF/Filter Paper Sandwiches,
7 x 8.5 cm, 20 pack
162-0219 Immun-Blot PVDF/Filter Paper Sandwiches,
7 x 8.5 cm, 50 pack
162-0216 Sequi-Blot PVDF/Filter Paper Sandwiches,
7 x 8.5 cm, 20 pack
162-0217 Sequi-Blot PVDF/Filter Paper Sandwiches,
7 x 8.5 cm, 50 pack
Criterion Membrane/Filter Paper Sandwiches
162-0232 0.2 μm Nitrocellulose/Filter Paper Sandwiches,
8.5 x 13.5 cm, 20 pack
162-0233 0.2 μm Nitrocellulose/Filter Paper Sandwiches,
8.5 x 13.5 cm, 50 pack
162-0234 0.45 μm Nitrocellulose/Filter Paper Sandwiches,
8.5 x 13.5 cm, 20 pack
162-0235 0.45 μm Nitrocellulose/Filter Paper Sandwiches,
8.5 x 13.5 cm, 50 pack
162-0262 Immun-Blot LF PVDF/Filter Paper,
8.5 x 13.5 cm, 10 pack
162-0263 Immun-Blot LF PVDF/Filter Paper,
8.5 x 13.5 cm, 20 pack
162-0238Immun-Blot PVDF/Filter Paper Sandwiches,
8.5 x 13.5 cm, 20 pack
162-0239 Immun-Blot PVDF/Filter Paper Sandwiches,
8.5 x 13.5 cm, 50 pack
162-0236 Sequi-Blot PVDF/Filter Paper Sandwiches,
8.5 x 13.5 cm, 20 pack
162-0237 Sequi-Blot PVDF/Filter Paper Sandwiches,
8.5 x 13.5 cm, 50 pack
Filter Paper
Blot Absorbent Filter Paper (Extra Thick)
170-3965 Extra Thick Blot Paper, 7.5 x 10 cm, for
Mini-PROTEAN gels, 60 sheets
170-3966Extra Thick Blot Paper, 7 x 8.4 cm, for
Mini-PROTEAN gels, 60 sheets
170-3967 Extra Thick Blot Paper, 8 x 13.5 cm, for Criterion
precast gels, 60 sheets
170-3968 Extra Thick Blot Paper, 14 x 16 cm, for PROTEAN
II xi gels, 30 sheets
170-3969 Extra Thick Blot Paper, 19 x 18.5 cm, for
PROTEAN II XL gels, 30 sheets
170-3958 Extra Thick Blot Paper, 10 x 15 cm, 30 sheets
170-3959 Extra Thick Blot Paper, 15 x 15 cm, 30 sheets
170-3960 Extra Thick Blot Paper, 15 x 20 cm, 30 sheets
Blot Absorbent Filter Paper (Thick)
170-3932 Thick Blot Paper, 7.5 x 10 cm, for Mini Trans-Blot
cassette, 50 sheets
170-4085 Thick Blot Paper, 9.5 x 15.2 cm, for Criterion
blotter, 50 sheets
170-3955 Thick Blot Paper, 14 x 16 cm, for PROTEAN II xi
gels, 25 sheets
170-3956 Thick Blot Paper, 15 x 20 cm, for Trans-Blot
cassette, 25 sheets
165-0921 Thick Blot Paper, 18 x 34 cm, for Model 224, 443,
and 543 slab gel dryers, 25 sheets
162-0161Bio-Dot/Bio-Dot SF Filter Paper, 7.7 x 11.3 cm,
60 sheets
165-0962 Filter Paper Backing, 35 x 45 cm, for stained gels,
25 sheets
Blot Absorbent Filter Paper (Thin)
162-0118 Thin Blot Paper, 33 cm x 3 m, 1 roll
Buffer Reagents
Electrophoresis Buffer Reagents
161-0610 Dithiothreitol (DTT), 1 g
161-0611 Dithiothreitol (DTT), 5 g
161-0729 EDTA, 500 g
170-6537 Gelatin, EIA grade, 200 g
161-0717 Glycine, 250 g
161-0718 Glycine, 1 kg
161-0724 Glycine, 2 kg
163-2109 Iodoacetamide, 30 g
161-0710 2-Mercaptoethanol, 25 ml
163-2101 Tributylphosphine (TBP), 200 mM, 0.6 ml
161-0713 Tricine, 500 g
161-0716 Tris, 500 g
161-0719 Tris, 1 kg
161-0730 Urea, 250 g
161-0731 Urea, 1 kg
Premixed Buffers
Electrophoresis Buffers
161-0732 10x Tris/Glycine/SDS, 1 L
161-0772 10x Tris/Glycine/SDS, 5 L cube
161-0734 10x Tris/Glycine, 1 L
161-0771 10x Tris/Glycine, 5 L cube
Blot Transfer
161-0734 161-0771 161-0778 161-0774 161-0775 161-0780 170-6435 and Processing Buffers
10x Tris/Glycine, 1 L
10x Tris/Glycine, 5 L cube
10x Tris/CAPS, 1 L
20x SSC, 1 L
20x SSC, 5 L cube
10x Phosphate Buffered Saline, 1 L
10x Tris Buffered Saline, 1 L
Detergents/Blocking Reagents
170-6537 Gelatin, EIA grade, 200 g
170-6404 Blotting-Grade Blocker, nonfat dry milk, 300 g
170-6531 Tween 20, EIA grade, 100 ml
161-0781 10% (w/v) Tween 20, for easy pipetting, 1 L
161-0418 SDS Solution, 20% (w/v), 1 L
161-0783 1x Phosphate Buffered Saline With 1% Casein, 1 L
161-0782 1x Tris Buffered Saline With 1% Casein, 1 L
Blotting Standards
161-0376 Precision Plus Protein™ WesternC™ Standards,
250 µl, 50 applications
161-0385 Precision Plus Protein WesternC Pack, includes
50 applications of WesternC standards and
50 applications of StrepTactin-HRP
161-0363 Precision Plus Protein Unstained Standards, 1 ml,
100 applications
161-0374 Precision Plus Protein Dual Color Standards, 500 μl
161-0394 Precision Plus Protein Dual Color Standards Value
Pack, 2.5 ml, 250 applications
161-0377 Precision Plus Protein Dual Xtra Standards,
500 µl, 50 applications
161-0375 Precision Plus Protein™ Kaleidoscope™
Standards, 500 μl
161-0395 Precision Plus Protein Kaleidoscope Standards
Value Pack, 2.5 ml, 250 applications
161-0373 Precision Plus Protein All Blue Standards, 500 μl
161-0393 Precision Plus Protein All Blue Standards Value
Pack, 2.5 ml, 250 applications
161-0305 Prestained SDS-PAGE Standards,
low range, 500 μl
161-0309 Prestained SDS-PAGE Standards,
high range, 500 μl
161-0318 Prestained SDS-PAGE Standards,
broad range, 500 μl
161-0324 Kaleidoscope™ Prestained Standards,
broad range, 500 μl
161-0304 SDS-PAGE Standards, low range, 200 μl
161-0303 SDS-PAGE Standards, high range, 200 μl
161-0317 SDS-PAGE Standards, broad range, 200 μl
161-0326 SDS-PAGE Standards, polypeptide, 200 μl
Accessory Reagents
170-6528 Avidin-HRP, 2 ml
170-6533 Avidin-AP, 1 ml
StrepTactin Conjugates
161-0380 Precision Protein™ StrepTactin-HRP Conjugate,
0.3 ml, 150 applications
161-0382 Precision Protein StrepTactin-AP Conjugate, 0.3 ml,
150 applications
Detection Reagents
Total Protein Stains
170-3127 SYPRO Ruby Protein Blot Stain, 200 ml
161-0400 Coomassie Brilliant Blue R-250, 10 g
170-6527 Colloidal Gold Total Protein Stain, 500 ml
Colorimetric Immun-Blot AP Assay Kits, with BCIP/NBT
170-6460 Goat Anti-Rabbit IgG (H + L)-AP Assay Kit
170-6461 Goat Anti-Mouse IgG (H + L)-AP Assay Kit
170-6462 Goat Anti-Human IgG (H + L)-AP Assay Kit
Immun-Blot AP kits include 0.5 ml blotting-grade conjugate, blottinggrade TBS, Tween 20, gelatin, and BCIP and NBT susbstrate solution.
Colorimetric Immun-Blot HRP Assay Kits, with 4CN
170-6463 Goat Anti-Rabbit IgG (H + L)-HRP Assay Kit
170-6464 Goat Anti-Mouse IgG (H + L)-HRP Assay Kit
170-6465 Goat Anti-Human IgG (H + L)-HRP Assay Kit
170-8235 Opti-4CN™ Substrate Kit
170-8237 Opti-4CN Goat Anti-Mouse Detection Kit
170-8238 Amplified Opti-4CN Substrate Kit
170-8240 Amplified Opti-4CN Goat Anti-Mouse Detection Kit
170-8239 Amplified Opti-4CN Goat Anti-Rabbit Detection Kit
Immun-Blot HRP kits include 0.5 ml blotting-grade conjugate,
blotting-grade TBS, Tween 20, gelatin, and 4CN susbstrate solution.
Blotting-Grade Conjugates, AP
170-6518 Goat Anti-Rabbit IgG-AP, 1 ml
170-6520 Goat Anti-Mouse IgG-AP, 1 ml
170-6521 Goat Anti-Human IgG-AP, 1 ml
Blotting-Grade Conjugates, HRP
170-6515 Goat Anti-Rabbit IgG (H + L)-HRP, 2 ml
170-6516 Goat Anti-Mouse IgG (H + L)-HRP, 2 ml
172-1050 Goat Anti-Human IgG (H + L)-HRP, 2 ml
170-6522 Protein A-HRP, 1 ml
170-6425 Protein G-HRP, 1 ml
170-6528 Avidin-HRP, 2 ml
Blotting Substrate Reagents
170-6432 AP Conjugate Substrate Kit, contains premixed
BCIP and NBT solutions, color development
buffer; makes 1 L color development solution
170-6539 AP Color Development Reagent, BCIP, 300 mg
(reagent necessary for purple color development;
also order #170-6532)
170-6532 AP Color Development Reagent, 5 NBT, 600 mg
(reagent necessary for purple color development,
also order #170-6539)
83
Ordering Info
Protein Blotting Guide
170-6431 HRP Conjugate Substrate Kit, contains
premixed 4CN, hydrogen peroxide solutions,
color development buffer; makes 1 L color
development solution
170-6534 HRP Color Development Reagent, 4CN, 5 g
170-6535 HRP Color Development Reagent, DAB, 5 g
™
Immun-Star AP Chemiluminescence Kits
170-5010 Goat Anti-Mouse-AP Detection Kit, includes
substrate, enhancer, antibody
170-5011 Goat Anti-Rabbit-AP Detection Kit, includes
substrate, enhancer, antibody
170-5012 AP Substrate Pack, includes substrate, enhancer
170-5018 AP Substrate, 125 ml
170-9450PharosFX System, PC or Mac, 110–240 V, includes
Quantity One software, sample tray set, fluorescence
filters (#170-7866, 170-7896), USB2 cable
170-9400Personal Molecular Imager (PMI) System, PC or Mac,
100/240 V, includes Quantity One software, sample
tray set, USB2 cable
Coomassie is a trademark of BASF Aktiengesellschaft. Nonidet is a trademark of Shell International Petroleum Co. Qdot is a trademark
of Life Technologies Corporation. SYPRO is a trademark of Invitrogen Corporation. StrepTactin and Strep-tag are trademarks of the
Institut für Bioanalytik GmbH. Tween is a trademark of ICI Americas Inc.
Precision Plus Protein standards are sold under license from Life Technologies Corporation, Carlsbad, CA for use only by the buyer
of the product. The buyer is not authorized to sell or resell this product or its components.
Strep-tag technology for western blot detection is covered by U.S. Patent Number 5,506,121 and by UK Patent Number 2,272,698.
StrepTactin is covered by German patent application P 19641876.3. Bio-Rad Laboratories, Inc. is licensed by Institut für Bioanalytik
GmbH to sell these products for research use only.
Each kit contains enough reagent for 2,500 cm2 of membrane, or
approximately 50 mini blots.
Immun-Star HRP Chemiluminescence Kits
170-5044 Goat Anti-Mouse-HRP Detection Kit, includes
complete reagents, 500 ml
170-5045 Goat Anti-Rabbit-HRP Detection Kit, includes
complete reagents, 500 ml
170-5043 Goat Anti-Mouse-HRP Detection Reagents,
include substrate, antibody, 500 ml
170-5042 Goat Anti-Rabbit-HRP Detection Reagents,
include substrate, antibody, 500 ml
170-5040 HRP Substrate, 500 ml
170-5041 HRP Substrate, 100 ml
170-5047 Goat Anti-Mouse-HRP Conjugate, 2 ml
170-5046 Goat Anti-Rabbit-HRP Conjugate, 2 ml
TABLE OF CONTENTS
500 ml substrate provides enough reagents for 4,000 cm2 of
membrane; 100 ml substrate provides enough for 800 cm2 of
membrane.
Detection Accessories
Mini Incubation Trays
170-3902 Mini Incubation Trays, 20 trays
170-3903 Mini Incubation Trays, 100 trays
Mini-PROTEAN II Multiscreen Apparatus
170-4017 Mini-PROTEAN II Multiscreen Apparatus, includes
2 sample templates, 2 gaskets, base plate
170-4018 Multiscreen Gaskets, 2 gaskets
Documentation Systems
170-7983 GS-800™ USB Calibrated Densitometer, PC or Mac,
100–240 V
170-8195Gel Doc™ XR+ System with Image Lab™ Software,
PC or Mac, includes darkroom, UV transilluminator,
epi-white illumination, camera, cables, Image
Lab software
170-8270Gel Doc EZ System with Image Lab Software, PC
or Mac, includes darkroom, camera, cables, Image
Lab software; samples trays (#170-8271, 170-8272,
170-8273, or 170-8274) are sold separately; sample
trays are required to use the system
170-8265 ChemiDoc™ XRS+ System With Image Lab Software,
PC or Mac, includes darkroom, UV transilluminator,
epi-white illumination, camera, power supply, cables,
Image Lab software
170-8640 VersaDoc™ MP 4000 System, PC or Mac,
100–240 V, includes CCD camera, darkroom,
power supply, cables, epi-illuminator,
transilluminator, fluorescence reference plate,
focusing target, Quantity One® software
170-8650 VersaDoc MP 5000 System, PC or Mac,
100–240 V, includes CCD camera, darkroom,
power supply, cables, epi-illuminator,
transilluminator, fluorescence reference plate,
focusing target, Quantity One software
170-9460PharosFX™ Plus System, PC or Mac, 110–240 V,
includes Quantity One software, sample tray set,
fluorescence filters (#170-7866, 170-7896)
and phosphor imaging filters, USB2 cable
Bio-Rad
Laboratories, Inc.
Bulletin 2895 Rev B
84
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