NuPAGE® Technical Guide

NuPAGE® Technical Guide
Instruction Manual
NuPAGE® Technical Guide
General information and protocols for using the
®
NuPAGE electrophoresis system
Version E
October 1, 2003
IM-1001
2
Table of Contents
Table of Contents ...........................................................................................................3
General Information........................................................................................................5
NuPAGE® Gel Specifications .........................................................................................6
Description of the NuPAGE® Electrophoresis System....................................................7
Preparing Samples.......................................................................................................11
Preparing Running Buffer.............................................................................................14
Electrophoresis of NuPAGE® Gels ...............................................................................17
Silver Staining ..............................................................................................................20
Coomassie® Staining....................................................................................................24
Gel Drying ....................................................................................................................28
Western Blotting ...........................................................................................................31
Using ZOOM® Gels ......................................................................................................37
Calibrating Protein Molecular Weight ...........................................................................39
Troubleshooting............................................................................................................42
Accessory Products......................................................................................................45
Recipes ........................................................................................................................47
Technical Service .........................................................................................................51
Purchaser Notification ..................................................................................................53
References...................................................................................................................54
3
4
General Information
Purpose of the
Guide
The NuPAGE® Technical Guide contains information about the NuPAGE®
Electrophoresis System and is intended to supplement the NuPAGE® Bis-Tris
Gel Instruction Card (IM-8042) and the NuPAGE® Tris-Acetate Gel Instruction
Card (IM-1025). Complete protocols for sample preparation, buffer preparation,
electrophoresis, staining, and blotting are provided in this guide.
To request the Instruction Cards or for additional information, contact Technical
Service (see page 51) or you may download the manuals from our web site at
www.invitrogen.com.
For description of the NuPAGE® electrophoresis system, see page 7.
Storage and Shelf
life
Store NuPAGE® Novex Bis-Tris Gels at 4-25°C and NuPAGE® Novex TrisAcetate Gels at +4°C.
The NuPAGE® Novex Bis-Tris Gels have a shelf life of 12 months when stored
at 4-25°C.
The NuPAGE® Novex Tris-Acetate Gels have a shelf life of 8 months when
stored at 4°C.
Do not freeze NuPAGE® Gels.
Using expired gels or improperly stored gels may result in poor band
resolution.
Handling the Gels
Gels are individually packaged in clear pouches with 10 ml of Packaging
Buffer. The Packaging Buffer contains low levels of residual acrylamide
monomer and 0.02% sodium azide. Gloves should be worn at all time when
handling gels.
Warning: This product contains a chemical (acrylamide) known to the state of
California to cause cancer. To obtain a MSDS, see page 51.
5
NuPAGE® Gel Specifications
Specifications
Recommended
Loading Volumes
Gel Matrix:
Acrylamide/Bisacrylamide
Gel Thickness:
1.0 mm
Gel Size:
8 cm x 8 cm
Cassette Size:
10 cm x 10 cm
Cassette Material:
Styrene Copolymer (recycle code 7)
Sample Well Configuration:
1, 9, 10, 12, 15, 17-well, 2D-well, and IPG well
The recommended loading volumes and protein load per band by the detection
method are provided in the table below.
Note: The 9- and 17-wells are compatible with any eight-channel pipette used
for loading samples from 96-well plates. An additional lane is included for
loading protein molecular weight standard.
Well Types
1 well
2D well
IPG well
9 well
10 well
12 well
15 well
17 well
6
Maximum Load
Volume
Coomassie® Staining
Silver Staining
Immunoblotting
700 µl
12 µg/band
1.0 mm
1.5 mm
400 µl
600 µl
12 µg/band
Scale your sample
load for the
sensitivity of your
silver staining kit.
1.0 mm
7 cm IPG Strip
N/A
Scale your
sample load
according to the
sensitivity of
your detection
method.
1.0 mm
28 µl
0.5 µg/band
1.0 mm
1.5 mm
25 µl
37 µl
0.5 µg/band
1.0 mm
20 µl
0.5 µg/band
1.0 mm
1.5 mm
15 µl
25 µl
0.5 µg/band
1.0 mm
15 µl
0.5 µg/band
1.0 mm
Maximum Protein Load Per Band by Detection Method
For use with the
SilverQuest™ or
SilverXpress ® Silver
Staining Kits, we
recommend a
protein load of
1 ng/band.
Description of the NuPAGE® Electrophoresis System
Introduction
The NuPAGE® Bis-Tris Electrophoresis System is a revolutionary neutral pH,
discontinuous SDS-PAGE, pre-cast polyacrylamide mini-gel system. The neutral
pH 7.0 environment during electrophoresis results in maximum stability of both
proteins and gel matrix, providing better band resolution than other gel systems
(see Advantages of the NuPAGE® Electrophoresis System, below).
The Laemmli system is the most widely used SDS-PAGE method for separating
a broad range of proteins (Laemmli, 1970). The highly alkaline operating pH of
the Laemmli system may cause band distortion, loss of resolution, or artifact
bands. The major causes of poor band resolution with the Laemmli system are:
Advantages of the
NuPAGE®
Electrophoresis
System
NuPAGE®
Electrophoresis
System
Components
•
Hydrolysis of polyacrylamide at the high gel casting pH of 8.7 resulting in a
short shelf life of 4-6 weeks
•
Chemical modifications such as deamination and alkylation of proteins due
to the high pH (9.5) of the separating gel
•
Reoxidation of reduced disulfides from cysteine containing proteins as the
redox state of the gel is not constant
•
Cleavage of Asp-Pro bond of the proteins when heated at 100°C in the
Laemmli sample buffer, pH 5.2 (Kubo, 1995).
The operating neutral pH of the NuPAGE® Gels and buffers provide following
advantages over the Laemmli system:
•
Longer shelf life of 8-12 months due to improved gel stability (see page 5)
•
Improved protein stability during electrophoresis at neutral pH resulting in
sharper band resolution and accurate results (Moos et al, 1998)
•
Complete reduction of disulfides under mild heating conditions (70°C for
10 minutes) and absence of cleavage of asp-pro bonds using the NuPAGE®
LDS Sample buffer (pH > 7.0 at 70°C)
•
Reduced state of the proteins maintained during electrophoresis and
blotting of the proteins by the NuPAGE® Antioxidant
The NuPAGE® Electrophoresis System consists of:
• NuPAGE® Novex Bis-Tris [Bis (2-hydroxyethyl) imino-tris (hydroxymethyl)
methane-HCl] Pre-Cast Gels for separating small to mid-size molecular weight
proteins
• NuPAGE® Novex Tris-Acetate Pre-Cast Gels for separating large molecular weight
proteins
• NuPAGE® LDS (Lithium dodecyl sulfate) Sample Buffer
• NuPAGE® Reducing Agent
• NuPAGE® Antioxidant
• NuPAGE® MES [2-(N-morpholino) ethane sulfonic acid] SDS or MOPS [3-(Nmorpholino) propane sulfonic acid] SDS Running Buffer for NuPAGE® Novex BisTris Gels
• NuPAGE® Tris-Acetate SDS Running Buffer for NuPAGE® Novex Tris- Acetate
Gels
•
NuPAGE® Transfer Buffer for blotting of NuPAGE® Novex Pre-Cast Gels
Continued on next page
7
Description of the NuPAGE® Electrophoresis System, Continued
NuPAGE® Bis-Tris
Discontinuous
Buffer System
NuPAGE® TrisAcetate
Discontinuous
Buffer System
Separation Range
of Proteins
The NuPAGE® Bis-Tris discontinuous buffer system involves three ions:
•
Chloride (-) is supplied by the gel buffer and serves as a leading ion due to
its high affinity to the anode as compared to other anions in the system. The
gel buffer ions are Bis-Tris+ and Cl- (pH 6.4).
•
MES or MOPS (-) serves as the trailing ion. The running buffer ions are Tris+,
MOPS-/MES-, and dodecylsulfate (-) (pH 7.3-7.7).
•
Bis-Tris (+) is the common ion present in the gel buffer and running buffer.
The combination of a lower pH gel buffer (pH 6.4) and running buffer (pH
7.3-7.7) results in a significantly lower operating pH of 7 during
electrophoresis.
The NuPAGE® Tris-Acetate discontinuous buffer system involves three ions:
•
Acetate (-) is supplied by the gel buffer and serves as a leading ion due to
its high affinity to the anode as compared to other anions in the system.
The gel buffer ions are Tris+ and Acetate- (pH 7.0).
•
Tricine (-) serves as the trailing ion from the running buffer. The running
buffer ions are Tris+, Tricine-, and dodecylsulfate (-) (pH 8.3).
•
Tris (+) is the common ion present in the gel buffer and running buffer. The
Tris-Acetate system also operates at a significantly lower operating pH of
8.1 during electrophoresis.
The NuPAGE® Gels have a wider range of separation on a single gel and also
separate proteins evenly throughout the low and high molecular weight ranges
than existing gels. Due to these advantages, most proteins are well resolved on
one of the five NuPAGE® gels (see Types of NuPAGE® Gels, next page).
By combining any of the NuPAGE® Novex Bis-Tris Gels with the MES SDS or
MOPS SDS Running Buffer, you can obtain six separation ranges for resolving
proteins over a wide molecular weight range of 1-200 kDa. The NuPAGE®
Novex Tris-Acetate gels resolve proteins in the molecular weight range of 36-400
kDa.
To choose the correct NuPAGE® Gel for your application, refer to the Gel
Migration Chart on our Web site at www.invitrogen.com or the catalog.
Continued on next page
8
Description of the NuPAGE® Electrophoresis System, Continued
Types of
NuPAGE® Gels
The NuPAGE® Novex Pre-Cast Gels are available in different acrylamide
concentrations, gel thickness, and well formats (see the table below).
NuPAGE® Novex Bis-Tris Gels
NuPAGE® Novex Tris-Acetate Gels
Separating Gel Acrylamide Concentration
10%
3-8%
12%
7%
4-12%
Stacking Gel Acrylamide Concentration
4%
3.2%
Gel Thickness
1.0 mm
1.0 mm
1.5 mm
1.5 mm
Well Format
1, 9, 10, 12, 15, 17, 2D, and IPG well
Formulation
10, 12, 15, and 2D well
The formulation for the NuPAGE® Gels is listed below:
NuPAGE® Novex Bis-Tris Gels
NuPAGE® Novex Tris-Acetate Gels
Bis-Tris-HCl buffer (pH 6.4)
Tris base
Acrylamide
Acetic acid
Bis-acrylamide
Acrylamide
Ammonium persulfate (APS)
Bis-acrylamide
Ultrapure water
TEMED
The separating gel operates at pH 7.0.
Ammonium persulfate (APS)
Ultrapure water
The separating gel operates at pH 8.1
The NuPAGE® Gels do not contain SDS. However, they are designed for
performing denaturing gel electrophoresis (see Applications, next page).
Crosslinker
The crosslinker concentration for the NuPAGE® Novex Pre-Cast Gel ranges
from 3.8-5% depending on the region of the gel.
Compatibility
The size of a NuPAGE® Novex Pre-Cast Gel is 10 x 10 cm (gel size is 8 x 8 cm).
We recommend using the XCell SureLock™ Mini-Cell (see page 45 for ordering
information) for the electrophoresis of NuPAGE® Novex Pre-Cast Gels to
obtain optimal and consistent performance.
Continued on next page
9
Description of the NuPAGE® Electrophoresis System, Continued
Staining NuPAGE®
Gels
The NuPAGE® Novex Pre-Cast Gels are compatible with most silver staining
protocols. We recommend using the SilverQuest™ Silver Staining Kit or the
SilverXpress® Silver Staining Kit (see pages 20-23) for silver staining of
NuPAGE® Gels.
The NuPAGE® Novex Pre-Cast Gels are compatible with any of the standard
Coomassie staining procedures. The protocols that are accelerated by heat are
preferable as the heat serves as a “fix” for proteins, especially smaller peptides.
The SimplyBlue™ SafeStain and Novex® Colloidal Coomassie Blue Staining Kit
(see pages 24-27) are recommended for staining NuPAGE® Gels.
The NuPAGE® Novex Pre-Cast Gels are also compatible with copper or zinc
staining.
Applications
The NuPAGE® Novex Pre-Cast Gels are used:
•
For separating proteins under denaturing conditions (NuPAGE® Bis-Tris
Gels and NuPAGE® Tris-Acetate Gels)
•
For separating proteins under non-denaturing (native) conditions
(NuPAGE® Tris-Acetate Gels).
•
For protein sequencing using Edman sequencing (from gels or PVDF)
Note: Do not use the NuPAGE® Bis-Tris Gels with NuPAGE® MOPS or MES
Running Buffer without SDS for native gel electrophoresis. This buffer system
may generate excessive heat resulting in poor band resolution. The protein of
interest may not migrate very well in a neutral pH environment if it is not
charged.
10
Preparing Samples
Introduction
General information on the sample buffer and reducing agent is provided
below. For sample preparation protocols, see page 13.
NuPAGE® LDS
Sample Buffer
Use the NuPAGE® LDS Sample Buffer (4X) for preparing samples for denaturing
gel electrophoresis with the NuPAGE® Gels.
For native gel electrophoresis with NuPAGE® Tris-Acetate Gels, use the Novex®
Tris-Glycine Native Sample Buffer (2X).
For optimal sample preparation in all SDS-PAGE protocols, including the
NuPAGE® system, denature and reduce the protein disulfide bonds under
slightly alkaline pH conditions. Since the pH of the NuPAGE® LDS Sample
Buffer is 8.4, sample reduction at this pH allows for maximal activity of the
reducing agent.
The NuPAGE® LDS Sample Buffer is a 4X concentrated solution containing
twice as much LDS as the 2X concentration of Novex® Tris-Glycine SDS or
Tricine SDS Sample Buffer. This makes the NuPAGE® LDS Sample Buffer more
viscous and difficult to pipet as compared to the Novex® Tris-Glycine or Tricine
Buffers.
The presence of more glycerol also increases the viscosity of the NuPAGE® LDS
Sample Buffer. By bringing the NuPAGE® LDS Sample Buffer to room
temperature (25°C), the buffer is more manageable.
Tracking Dye
The NuPAGE® LDS Sample Buffer contains Coomassie G250 and Phenol Red as
tracking dyes instead of bromophenol blue.
Coomassie G250 gives a sharp dye front with both MES and MOPS SDS
Running Buffers and migrates much closer to the moving ion front than
bromophenol blue. This ensures that small peptides do not run off the gel.
Bromophenol blue runs more slowly than some peptides with the MES SDS
Running Buffer.
The concentration of the tracking dye (Coomassie G250) is increased in the
NuPAGE® LDS Sample Buffer to enhance viewing of the dye front.
Reducing Agent
The NuPAGE® Reducing Agent contains 500 mM dithiothreitol (DTT) at a 10X
concentration and is available in a ready-to-use, stabilized liquid form (see page 45
for ordering information). Use the NuPAGE® Reducing Agent to prepare samples
for reducing gel electrophoresis.
β-mercaptoethanol is compatible with the NuPAGE® system and can be used with
the NuPAGE® gels at a final concentration of 2.5%. Choice of the reducing agent is a
matter of preference and either DTT or β-mercaptoethanol can be used. We
recommend adding the reducing agent to the sample within an hour of loading
the gel.
Avoid storing reduced samples for long periods even if they are frozen. This will
result in the reoxidation of samples during storage and produce inconsistent results.
Continued on next page
11
Preparing Samples, Continued
Important
Running Reduced
and Non-Reduced
Samples
Materials Supplied
by the User
Do not use the NuPAGE® Antioxidant as a sample reducing agent. The
antioxidant is not efficient in reducing the disulfide bonds. This will result in
partially reduced bands with substantial background smearing in the lane.
The antioxidant maintains the sample proteins that have been previously
reduced with a reducing agent in a reduced state and prevents the proteins
from reoxidizing during electrophoresis.
For optimal results, we do not recommend running reduced and non-reduced
samples on the same gel.
If you do choose to run reduced and non-reduced samples on the same gel,
follow these guidelines:
•
Do not run reduced and non-reduced samples in adjacent lanes. The
reducing agent may have a carry-over effect on the non-reduced samples if
they are in close proximity.
•
If you are running reduced and non-reduced samples on the same gel, omit
the antioxidant (see page 14). The antioxidant will have a deleterious effect
on the non-reduced samples. The bands will be sharper on NuPAGE® Gels
relative to other gel systems, even without the use of the antioxidant.
You will need the following items:
•
Protein sample
•
Deionized water
For denaturing electrophoresis
•
NuPAGE® LDS Sample Buffer (see page 45 for ordering information or
page 47 for a recipe)
•
NuPAGE® Reducing Agent (see page 45 for ordering information)
For non-denaturing electrophoresis
•
Novex® Tris-Glycine Native Sample Buffer (see page 45 for ordering
information or page 47 for a recipe)
Continued on next page
12
Preparing Samples, Continued
Preparing
Samples for
Denaturing
NuPAGE® Gel
Electrophoresis
Instructions are provided below to prepare reduced or non-reduced samples
for denaturing gel electrophoresis using the NuPAGE® Novex Bis-Tris or TrisAcetate Gels.
The NuPAGE® LDS Sample Buffer and NuPAGE® Reducing Agent are
available from Invitrogen (see page 45 for ordering information).
For reduced sample, add the reducing agent immediately prior to
electrophoresis to obtain the best results.
Reagent
Reduced Sample
Sample
x µl
NuPAGE® LDS Sample Buffer (4X)
2.5 µl
NuPAGE® Reducing Agent (10X)
1 µl
Deionized Water
to 6.5 µl
Total Volume
10 µl
Non-reduced Sample
x µl
2.5 µl
-to 7.5 µl
10 µl
See page 47 for a recipe of sample buffer, if you are preparing the sample
buffer.
Preparing
Samples for
Non-Denaturing
NuPAGE® Gel
Electrophoresis
Instructions are provided below to prepare samples for non-denaturing (native)
gel electrophoresis using the NuPAGE® Novex Tris-Acetate Gels.
The Novex® Tris-Glycine Native Sample Buffer is available from Invitrogen (see
page 45 for ordering information).
Reagent
Sample
Novex® Tris-Glycine Native Sample Buffer (2X)
Deionized Water
Total Volume
Volume
x µl
5 µl
to 5 µl
10 µl
See page 47 for a recipe of the sample buffer, if you are preparing the sample
buffer.
Heating Samples
Heat the sample for denaturing electrophoresis (reduced or non-reduced) at
70°C for 10 minutes for optimal results.
Do not heat samples for non-denaturing (native) electrophoresis.
13
Preparing Running Buffer
Introduction
General information on the running buffer and antioxidant is provided below.
Instructions for preparing running buffers for denaturing and non-denaturing
electrophoresis are provided on the next page.
NuPAGE® SDS
Running Buffer
Three types of NuPAGE® Running Buffers are used for denaturing gel
electrophoresis of NuPAGE® Gels. See page 45 for ordering information.
•
NuPAGE® MES SDS Running Buffer is used with NuPAGE® Novex Bis-Tris
Gels to resolve small molecular weight proteins
•
NuPAGE® MOPS SDS Running Buffer is used with NuPAGE® Novex BisTris Gels to resolve mid-size proteins
•
NuPAGE® Tris-Acetate SDS Running Buffer is used with NuPAGE® Novex
Tris-Acetate Gels to resolve high molecular weight proteins
The NuPAGE® MES SDS Running Buffer and NuPAGE® MOPS SDS Running
Buffers have different pKa’s, resulting in MES being a faster running buffer than
MOPS. The difference in ion migration affects the stacking and the separation
ranges of proteins with these buffers.
For native gel electrophoresis with NuPAGE® Novex Tris-Acetate Gels, use the
Novex® Tris-Glycine Native Running Buffer.
To prepare Running Buffers for electrophoresis, see next page.
NuPAGE®
Antioxidant
The reducing agents, DTT and β-mercaptoethanol, do not co-migrate through the
gel with the sample in a neutral pH environment of the NuPAGE® Gels. Instead,
the reducing agent tends to remain at the top of the gel and not migrate fully
throughout the gel. Disulfide bonds are less reactive at neutral pH and are less
likely to reoxidize than in a higher pH system. However, in the absence of an
antioxidant some reoxidization may occur during the electrophoresis, resulting
in slightly diffuse bands.
The NuPAGE® Antioxidant (a proprietary reagent) is added to the running
buffer in the upper (cathode) buffer chamber only when performing
electrophoresis under reducing conditions. The NuPAGE® Antioxidant migrates
with the proteins during electrophoresis preventing the proteins from
reoxidizing and maintaining the proteins in a reduced state. The NuPAGE®
Antioxidant also protects sensitive amino acids such as methionine and
tryptophan from oxidizing.
We also recommend using the NuPAGE® Antioxidant with reduced samples that
have been alkylated, for optimal results.
The NuPAGE® Antioxidant is NOT compatible with gel systems other than the
NuPAGE® system as the antioxidant is not efficient at higher pHs of other gel
systems.
Continued on next page
14
Preparing Running Buffer, Continued
Materials Supplied
by the User
You will need the following items:
•
Deionized water
For denaturing electrophoresis
•
NuPAGE® MES or MOPS SDS Running Buffer (see page 45 for ordering
information or page 47 for a recipe)
•
NuPAGE® Tris Acetate Running Buffer (see page 45 for ordering
information or page 47 for a recipe)
•
NuPAGE® Antioxidant
For non-denaturing electrophoresis
•
Preparing Buffer
for Denaturing
Electrophoresis
Novex® Tris-Glycine Native Running Buffer (see page 45 for ordering
information or page 47 for a recipe)
NuPAGE® SDS Running Buffer (20X) is available from Invitrogen (see page 45).
Reducing Conditions
1.
Prepare 1000 ml of 1X NuPAGE® SDS Running Buffer using NuPAGE® SDS
Running Buffer (20X) as follows:
NuPAGE® SDS Running Buffer (20X, MES, MOPS, or Tris-Acetate) 50 ml
Deionized Water
950 ml
Total Volume
1000 ml
2.
Mix thoroughly and set aside 800 ml of the 1X NuPAGE® SDS Running
Buffer for use in the Lower (Outer) Buffer Chamber of the XCell SureLock™
Mini-Cell.
3.
Immediately, prior to electrophoresis, add 500 µl of NuPAGE® Antioxidant
to 200 ml of 1X NuPAGE® SDS Running Buffer from Step 1 for use in the
Upper (Inner) Buffer Chamber of the XCell SureLock™ Mini-Cell. Mix
thoroughly.
Non-Reducing Conditions
1.
Prepare 1000 ml of 1X NuPAGE® SDS Running Buffer using NuPAGE® SDS
Running Buffer (20X) as follows:
NuPAGE® SDS Running Buffer (20X, MES or MOPS) 50 ml
Deionized Water
950 ml
Total Volume
1000 ml
2.
Mix thoroughly. Fill the Upper and Lower Buffer Chamber of the XCell
SureLock™ Mini-Cell with this Running Buffer.
See page 47 for a recipe of the NuPAGE® SDS Running Buffers, if you are
preparing the running buffers.
Continued on next page
15
Preparing Running Buffer, Continued
Preparing Buffer
for NonDenaturing
Electrophoresis
Novex® Tris-Glycine Native Running Buffer (10X) is available from Invitrogen
(see page 45).
1.
Prepare 1000 ml of 1X Native Running Buffer using the Novex® TrisGlycine Native Running Buffer (10X) as follows:
Novex® Tris-Glycine Native Running Buffer (10X)
Deionized Water
Total Volume
2.
100 ml
900 ml
1000 ml
Mix thoroughly and use 800 ml of this Running Buffer in the Lower and
Upper Buffer Chambers of the XCell SureLock™ Mini-Cell.
See page 47 for a recipe of the Novex® Tris-Glycine Native Running Buffer, if
you are preparing the running buffer.
16
•
If you forget to add the antioxidant to the upper buffer chamber, some
bands may be slightly fuzzier and more diffuse due to reoxidation of some
proteins during electrophoresis.
•
We recommend preparing the running buffer for the upper chamber with
the antioxidant within half an hour of planned use. If the antioxidant is
added to the running buffer too long before use, gels may exhibit signs of
reoxidation (slightly fuzzier bands).
•
If you have added 0.5 ml of antioxidant to the total amount of buffer (for
upper and lower buffer chamber) by accident, the required concentration of
antioxidant will be lower and the antioxidant will not be effective. If you
wish to add antioxidant to the total amount of buffer (for upper and lower
buffer chamber), add 2.5 ml of antioxidant to obtain the expected results.
However, this is not recommended as high current will be generated and
the antioxidant is wasted.
•
If you have switched the antioxidant and the reducing agent (used the
antioxidant in the sample buffer and the reducing agent in the running
buffer) by accident, the reducing agent (DTT) will not migrate into the gel
and the antioxidant will not effectively reduce the samples, resulting in
decreased staining sensitivity.
Electrophoresis of NuPAGE® Gels
Introduction
Instructions are provided below for electrophoresis of the NuPAGE® Gels using
the XCell SureLock™ Mini-Cell. For more information on the XCell SureLock™
Mini-Cell, refer to the manual (IM-9003). This manual is available on our Web
site at www.invitrogen.com or contact Technical Service (see page 51).
If you are using any other electrophoresis mini-cell, follow the manufacturer’s
recommendations.
Important
Procedure using
XCell SureLock™
Mini-Cell
To ensure success with the NuPAGE® Electrophoresis System, remember the
important points listed below:
•
Under NO circumstances should Tris-Glycine SDS buffers be used with
NuPAGE® Gels for any denaturing gel electrophoresis (see page 42 for the
outcome of your results using incorrect buffers)
•
Use ONLY NuPAGE® SDS buffers (see page 14)
•
DO NOT BOIL samples. Heat samples at 70°C for 10 minutes (see page 13)
•
Inner and Outer Buffer Chambers MUST be filled with the recommended
amount of running buffer to prevent excessive heating (see below).
Wear protective gloves and safety glasses when handling gels.
XCell SureLock™ Mini-Cell require 200 ml for the Upper Buffer Chamber and
600 ml for the Lower Buffer Chamber.
1.
Remove the NuPAGE® Gel from the pouch.
2.
Rinse the gel cassette with deionized water. Peel off the tape from the
bottom of the cassette.
3.
In one smooth motion, gently pull the comb out of the cassette.
4.
Rinse the sample wells with 1X NuPAGE® SDS Running Buffer. Invert
the gel and shake to remove the buffer. Repeat two more times.
5.
Orient the two gels in the Mini-Cell such that the notched “well” side of
the cassette faces inwards toward the Buffer Core. Seat the gels on the
bottom of the Mini-Cell and lock into place with the Gel Tension Wedge.
Refer to the XCell SureLock™ Mini-Cell manual (IM-9003) for detailed
instructions.
Note: If you are using only one gel, the plastic Buffer Dam replaces the second
gel cassette.
6.
Fill the Upper Buffer Chamber with a small amount of the running buffer
to check for tightness of seal. If you detect a leak from Upper to the
Lower Buffer Chamber, discard the buffer, reseal the chamber, and refill.
7.
Once the seal is tight, fill the Upper Buffer Chamber (inner) with the
appropriate 1X running buffer (see page 15 for running buffer
preparation). The buffer level must exceed the level of the wells.
Continued on next page
17
Electrophoresis of NuPAGE® Gels, Continued
Procedure using
XCell SureLock™
Mini-Cell,
continued
Electrophoresis
Conditions
Note: If you are running reduced samples, remember to fill the Upper Buffer
Chamber with 200 ml of running buffer containing the NuPAGE®
Antioxidant (see page 15).
8.
Load an appropriate volume of sample at the desired protein
concentration onto the gel (see page 6 for recommended loading
volumes).
9.
Load appropriate protein molecular weight markers (see page 45 for
ordering information).
10.
Fill the Lower (outer) Buffer Chamber with 600 ml of the appropriate 1X
running buffer.
Run your gels according to the following protocol:
Gel Type
®
NuPAGE Novex Bis-Tris Gels
with MES SDS Running Buffer
®
NuPAGE Novex Bis-Tris Gels
with MOPS SDS Running Buffer
®
NuPAGE Novex Tris-Acetate
Gels
®
NuPAGE Novex Tris-Acetate
Native Gels
Voltage
Expected Current*
Run Time
200 V constant†
Start: 110-125 mA/gel
35 minutes
End: 70-80 mA/gel
200 V constant†
Start: 100-115 mA/gel
50 minutes
End: 60-70 mA/gel
150 V constant
Start: 40-55 mA/gel
1 hour
End: 25-40 mA/gel
150 V constant
Start: 18 mA/gel
~2 hours
End: 7 mA/gel
Run times
may vary
† Recommended voltage for 9 and 17-well gels is 150-175 volts
Continued on next page
18
Electrophoresis of NuPAGE® Gels, Continued
Removing the Gel
after
electrophoresis
1.
After electrophoresis is complete, shut off the power, disconnect electrodes,
and remove gel(s) from the XCell SureLock™ Mini-Cell.
2.
Separate each of the three bonded sides of the cassette by inserting the Gel
Knife into the gap between the cassette’s two plates. The notched (“well”)
side of the cassette should face up.
3.
Push down gently on the knife handle to separate the plates. Repeat on
each side of the cassette until the plates are completely separated.
Caution: Use caution while inserting the gel knife between the two plates to
avoid excessive pressure towards the gel.
4.
Carefully remove and discard the top plate, allowing the gel to remain on
the bottom (slotted) plate.
5.
If blotting, proceed to the Western Transfer Protocol on page 31 without
removing the gel from the bottom plate.
6.
If staining, remove the gel from the plate by one of the methods:
7.
•
Use the sharp edge of the gel knife to remove the bottom lip of the gel.
The gel knife should be at a 90° angle, perpendicular to the gel and the
slotted half of the cassette. Push down on the knife, and then repeat the
motion across the gel to cut off the entire lip. Hold the plate and gel
over a container with the gel facing downward and use the knife to
carefully loosen one lower corner of the gel and allow the gel to peel
away from the plate.
•
Hold the plate and gel over a container with the gel facing downward.
Gently push the gel knife through the slot in the cassette, until the gel
peels away from the plate. Cut the lip off of the gel after fixing,
staining, but before drying.
Fix and stain the gel as described on pages 20-27.
19
Silver Staining
Introduction
Instructions are provided below for silver staining the NuPAGE® Gels using the
SilverQuest™ Silver Staining Kit and the SilverXpress® Silver Staining Kit (see
page 45 for ordering information).
If you are using any other silver staining kit, follow the manufacturer’s
recommendations.
The NuPAGE® system is more effective in reducing proteins and maintaining
proteins in their reduced state. This may cause any minor contaminants present
in the protein to be more visible under the sensitive silver staining techniques
with the NuPAGE® system than in other systems.
Materials Supplied
by the User
You will need following items for silver staining:
•
Staining container
•
Rotary Shaker
•
Ultrapure water (>18 megohm/cm resistance recommended)
•
Teflon coated stir bars
•
Disposable 10 ml pipettes
•
Clean glass bottles for reagent preparation
•
Graduated glass cylinders
•
Protein molecular weight markers (Mark 12™ Unstained Standard,
recommended; see page 45 for ordering information)
For SilverQuest™ Silver Staining
•
Ethanol
MEND
ION
AT
RECOM
• Fixative (40% ethanol, 10% acetic acid)
For SilverXpress® Silver Staining
•
Methanol
•
Acetic acid
For optimal silver staining results, follow these guidelines:
•
Be sure to wear rubber gloves that have been rinsed with deionized water
while handling gels
•
Use clean containers and designate these containers for silver staining
purposes only
•
Make sure the size of the container permits free movement of the gel
during shaking and complete immersion in solution while staining
•
Do not touch the gel with bare hands or metal objects and do not put
pressure on gels while handling or changing solutions
•
Use teflon coated stir bars and clean glass containers to prepare reagents
•
Avoid cross contamination of kit reagents
•
Use freshly made solutions
Continued on next page
20
Silver Staining, Continued
Preparing
Solutions for
SilverQuest™
Silver Staining
Use the reagents provided in the SilverQuest™ Silver Staining Kit to prepare the
following solutions for staining:
•
Sensitizing solution
Ethanol
30 ml
Sensitizer
10 ml
Ultrapure water
•
Staining solution
Stainer
Ultrapure water
•
to 100 ml
1 ml
to 100 ml
Developing solution
Developer
Developer enhancer
Ultrapure water
10 ml
1 drop
to 100 ml
Note: You may prepare all solutions immediately before starting the staining
protocol or prepare them as you proceed to the next step.
SilverQuest™
Microwave Silver
Staining Protocol
The microwave protocol for silver staining NuPAGE® Gels with SilverQuest™
Silver Staining Kit is provided below. For the Basic Protocol and more details on
the staining procedure, refer to the SilverQuest™ Silver Staining Kit Manual
(IM-6070). This manual is available on our Web site at www.invitrogen.com or
contact Technical Service (see page 51).
For use with an 8 x 8 cm NuPAGE® Gel, 1.0 mm thick. Use 100 ml of each
solution per gel.
Note: You may have to optimize the staining protocol, if the dimensions of your
gel are not the same as mentioned above.
1. After electrophoresis, place the gel in a clean microwaveable staining tray of
the appropriate size. Rinse the gel briefly with ultrapure water.
2. Place the gel in 100 ml of fixative and microwave at high power (700 watts)
for 30 seconds. Remove the gel from the microwave and gently agitate it for
5 minutes at room temperature. Decant the fixative.
3.
Wash the gel with 100 ml of 30% ethanol in a microwave at high power for
30 seconds. Remove the gel from the microwave and gently agitate it for
5 minutes at room temperature on a rotary shaker. Decant the ethanol.
Continued on next page
21
Silver Staining, Continued
SilverQuest™
Microwave Silver
Staining Protocol,
continued
Add 100 ml of Sensitizing solution to the washed gel. Microwave at high
power for 30 seconds. Remove the gel from the microwave and place it on a
rotary shaker for 2 minutes at room temperature. Decant the Sensitizing
solution.
5. Wash the gel twice in 100 ml ultrapure water. Microwave at high power for
30 seconds. At each wash step, remove the gel from the microwave and
gently agitate it for 2 minutes at room temperature.
6. Place the gel in 100 ml of Staining solution. Microwave at high power for
30 seconds. Remove the gel from the microwave and gently agitate it for
5 minutes at room temperature.
7. Decant the Staining solution and wash the gel with 100 ml of ultrapure
water for 20-60 seconds. Do not wash the gel for more than a minute.
8. Place the gel in 100 ml of Developing solution and incubate for 5 minutes at
room temperature with gentle agitation on a rotary shaker. Do not
microwave.
9. Once the desired band intensity is achieved, immediately add 10 ml of
Stopper directly to the gel still immersed in Developing solution and gently
agitate the gel for 10 minutes. The color changes from pink to clear
indicating that the end of development.
10. Wash the gel with 100 ml of ultrapure water for 10 minutes. For gel drying,
see page 28.
If you need to destain the gel for mass spectrometry analysis, see the
SilverQuest™ Silver Staining Kit Manual (IM-6070).
Preparing
Solutions for
SilverXpress®
Silver Staining
Prepare the reagents as described below. If you are staining two gels, double
the reagent volumes. Note: The final volumes of solutions containing methanol
and water reflect a volume shrinkage which occurs when these two reagents
are mixed. Do not adjust volumes of components or final volume.
4.
•
•
•
•
Fixing solution
Methanol
Acetic Acid
Ultrapure water
Sensitizing solution
Methanol
Sensitizer
Ultrapure water
Staining solution
Stainer A
Stainer B
Ultrapure water
Developing Solution
Developer
Ultrapure water
100 ml
20 ml
90 ml
100 ml
5 ml
105 ml
5 ml
5 ml
90 ml
5 ml
95 ml
Continued on next page
22
Silver Staining, Continued
SilverXpress®
Silver Staining
Protocol
The following staining procedure is for a 1 mm NuPAGE® Gel. If you are using
1.5 mm NuPAGE® Gel, double the incubation time.
Note: Gels may be stored in the second Sensitizing Solution overnight, if
desired. For gel drying, see page 28.
Step
1
2A
2B
3A
3B
Solution
Vol/Gel
NuPAGE® Gel Type
Tris –Acetate Gel
Bis-Tris Gel
Fix the gel in Fixing Solution.
200 ml
10 minutes
10 minutes
Decant the Fixing Solution
and incubate the gel in two
changes of Sensitizing
Solution.
100 ml
10 minutes
30 minutes
100 ml
10 minutes
30 minutes
200 ml
5 minutes
10 minutes
200 ml
5 minutes
10 minutes
Decant the Sensitizing
Solution and rinse the gel
twice with ultrapure water.
4
Incubate the gel in Staining
Solution.
100 ml
15 minutes
15 minutes
5A
Decant the Staining Solution
and rinse the gel twice with
ultrapure water.
200 ml
5 minutes
5 minutes
200 ml
5 minutes
5 minutes
5B
6
Incubate the gel in
Developing Solution.
100 ml
3-15 minutes
3-15
minutes
7
Add the Stopping Solution
directly to the gel when the
desired staining intensity is
reached.
5 ml
10 minutes
10 minutes
Decant the Stopping Solution
and wash the gel three times
in ultrapure water.
200 ml
10 minutes
10 minutes
200 ml
10 minutes
10 minutes
200 ml
10 minutes
10 minutes
8A
8B
8C
Molecular Weight
Calibration
Guidelines and apparent molecular weight values for the Novex® protein
molecular weight standards in the NuPAGE® buffer system is provided on
pages 39-41.
23
Coomassie® Staining
Introduction
Instructions are provided below for Coomassie® staining of NuPAGE® Gels
using the SimplyBlue™ SafeStain, Colloidal Blue Staining Kit, and Coomassie®
R-250 (see page 45 for ordering information).
If you are using any other coomassie® staining kit, follow the manufacturer’s
recommendations.
If you are staining low molecular weight peptides (< 2.5 kDa), we recommend
fixing the gel in 5% glutaraldehyde and 50% methanol for one hour and then
follow the instructions in the Colloidal Blue Staining Kit Manual (IM-6025) for
small peptides.
Materials Supplied
by the User
You will need the following items for staining:
•
Staining container
•
Ultrapure water or deionized water
•
Orbital Shaker
•
Protein molecular weight standards (see page 45 for ordering information)
•
Microwave oven and 20% NaCl (if using SimplyBlue™ SafeStain microwave
protocol, see page 25)
•
Methanol and acetic acid (if using Colloidal Blue Staining Kit, see page 26)
For Coomassie® R-250 staining
•
0.1% Coomassie® R-250 in 40% ethanol and 10% acetic acid
•
Destaining Solution (10% ethanol and 7.5% acetic acid)
Continued on next page
24
Coomassie® Staining, Continued
SimplyBlue™
SafeStain
Microwave
Protocol
The microwave protocol for staining NuPAGE® Gels with SimplyBlue™
SafeStain is provided below. For the Basic Protocol and more details on the
staining procedure, refer to the SimplyBlue™ SafeStain Manual (IM-6050). This
manual is available on our Web site at www.invitrogen.com or contact
Technical Service (see page 51).
The procedure is written for 1.0 mm mini-gels. For 1.5 mm mini-gels, use the
values in parentheses.
Caution: Use caution while using the stain in a microwave oven. Do not
overheat the staining solutions.
1.
After electrophoresis, place the gel in 100 ml of ultrapure water in a loosely
covered container and microwave on High (950 to 1100 watts) for 1 minute
until the solution almost boils.
2.
Gently shake the gel on an orbital shaker or rocker for 1 minute (2
minutes). Discard the water.
3.
Repeat Steps 1 and 2 two more times.
4.
Add 20 ml (30) ml of SimplyBlue™ SafeStain and microwave on High for
45 seconds to 1 minute (1.5 minutes) until the solution almost boils.
5.
Shake the gel on an orbital shaker or rocker for 5 minutes (10 minutes).
Detection limit: 20 ng BSA.
6.
Wash the gel in 100 ml of ultrapure water for 10 minutes on a shaker.
Detection limit: 10 ng BSA.
7.
Add 20 ml of 20% NaCl for at least 5 minutes. Detection limit: 5 ng BSA.
Gel can be kept for several weeks in the salt solution.
8.
For gel-drying, see page 28.
Continued on next page
25
Coomassie® Staining, Continued
Colloidal Blue
Staining Kit
Protocol
A brief staining protocol for staining NuPAGE® Gels with the Colloidal Blue
Staining Kit (see page 45 for ordering information) is provided below. For more
details on the staining procedure, refer to the Manual (IM-6025). This manual is
available on our Web site at www.invitrogen.com or contact Technical Service
(see page 51).
Colloidal Blue Staining Kit Protocol for NuPAGE® Novex Tris-Acetate Gels
1.
Prepare staining solution for a single gel consisting of 55 ml deionized
water, 20 ml methanol, 5 ml Stainer B and 20 ml Stainer A. Be sure to
shake Stainer B prior to making the solution.
Note: For two gels, double the volume of reagents used for staining.
2.
Incubate the gel in this staining solution for a minimum of 3 hours and a
maximum of 12 hours at room temperature with gentle shaking.
3.
Decant staining solution and add a minimum of 200 ml of deionized
water per gel to the staining container. Gently shake gel in water for at
least 7 hours. Gel will have a clear background after 7 hours in water.
4.
For gel-drying, see page 28.
Colloidal Blue Staining Kit Protocol for NuPAGE® Novex Bis-Tris Gels
Note: If you are staining low molecular weight peptides (< 2.5 kDa), we
recommend fixing the gel in 5% glutaraldehyde and 50% methanol for one
hour and then follow the instructions in the Colloidal Blue Staining Kit Manual
(IM-6025) for small peptides.
1.
Prepare fixing solution consisting of 50% methanol, 10% acetic acid, and
40% deionized water.
2.
Incubate the gel in the fixing solution for 10 minutes at room temperature
with gentle shaking.
3.
Prepare the staining solution containing 55 ml deionized water, 20 ml
methanol, and 20 ml Stainer for one gel.
4.
Incubate the gel in this staining solution for 10 minutes at room
temperature with gentle shaking.
5.
Add 5 ml Stainer B per gel to the staining solution from previous step.
Continue staining for a minimum of 3 hours and a maximum of 16 hours.
6.
Decant the staining solution and add 200 ml of deionized water per gel to
the staining container. Gently shake the gel in water for at least 7 hours.
Gel will have a clear background after 7 hours in water.
7.
For gel-drying, see page 28.
Note: NuPAGE® Gels can be left in deionized water for up to 3 days without
significant change in band intensity and background clarity.
For long-term storage (over 3 days), keep the gel in a 20% ammonium sulfate
solution at 4°C.
26
Coomassie® Staining, Continued
Coomassie® R-250
Microwave
Staining Protocol
The coomassie® staining protocol described below is recommended for staining
NuPAGE® Gels. You may use any Coomassie® staining protocol of choice.
1.
Prepare the staining solution containing 0.1% Coomassie® R-250 in
40% ethanol, 10% acetic acid.
2.
After electrophoresis, incubate 1 or 2 gels in a staining container containing
100 ml of staining solution prepared in Step 1.
3.
Loosely cover the staining container and heat in a microwave oven at full
power for 1 minute. To prevent hazardous, flammable vapors from
forming, do not allow the solution to boil.
4.
Remove the staining container from the microwave oven and gently shake
the gel for 15 minutes at room temperature on an orbital shaker.
5.
Decant the stain and rinse the gel once with deionized water.
6.
Prepare a destain solution containing 10% ethanol and 7.5% acetic acid.
7.
Place one or two stained gels in a staining container containing 100 ml of
destain solution prepared in Step 6.
8.
Loosely cover the staining container and heat in a microwave oven at full
power for 1 minute.
9.
Gently shake the gel at room temperature on an orbital shaker until the
desired background is achieved. Note: The NuPAGE® Gels destain faster
than other Novex® Gels. To prevent over destaining of NuPAGE® Gels if
destaining overnight, dilute the destain solution by adding 100 ml of
deionized water to 100 ml of the destain solution in the staining container.
10. For-gel-drying, see page 28.
Molecular Weight
Calibration
Guidelines and apparent molecular weight values for the Novex® protein
molecular weight standards in the NuPAGE® buffer system is provided on
pages 39-41.
27
Gel Drying
Introduction
Gels can be dried using passive evaporation (air-drying) or vacuum drying.
Vacuum drying is faster than passive air-drying methods but often result in
cracked gels due to the speed of dehydration.
We recommend drying Novex® Pre-Cast gels using passive air-drying methods
such as DryEase® Mini-Gel Drying System (see page 45 for ordering
information). If certain applications require drying the gel using vacuum
drying, follow the recommendations on page 30 to minimize cracking of
vacuum dried gels.
Do not leave colloidal Coomassie® stained gels in Gel-Dry™ solution (or any
equilibration solution which contains > 20% alcohol) for more than 5 minutes.
Gels left in this solution for longer than 5 minutes will lose band intensity and
detection limits may increase.
Materials Supplied
by the User
DryEase® Mini-Gel
Drying System
You will need the following items. Ordering information is provided on page 45.
•
DryEase® Mini-Gel Drying System
•
Gel-Dry™ Drying Solution (or prepare your own gel drying solution
containing 30% methanol and 5% glycerol)
•
StainEase® Gel Staining Tray
A brief gel drying protocol using the DryEase® Mini-Gel Drying System is
provided below. For more details on this system, refer to the DryEase® MiniGel Drying System manual (IM-2380). This manual is available for
downloading from our Web site at www.invitrogen.com or contact Technical
Service (see page 51).
Wear gloves while handling gels and gel drying solution.
1.
After all staining and destaining steps are complete, wash the destained
gel(s) three times for two minutes each time in deionized water (50 ml per
mini-gel) on a rotary shaker.
2.
Decant the water and add fresh Gel-Dry™ Drying Solution (35 ml per minigel).
3.
Equilibrate the gel in the Gel-Dry™ Drying Solution by shaking the gel for
15-20 minutes in the StainEase® Gel Staining Tray or in a round container.
Note: Do not equilibrate coomassie® stained gels in the Gel-Dry™ Drying
Solution for more than 5 minutes to avoid losing band intensity.
4.
Cut any rough edges off the gel (including the wells and the gel foot) using
the Gel Knife or a razor blade.
5.
Remove 2 sheets of cellophane per gel from the package.
6.
Immerse one sheet of cellophane in the Gel-Dry™ Drying Solution. Allow
15-20 seconds for complete wetting before adding additional sheets. Do not
soak the cellophane sheets for more than 2 minutes.
Continued on next page
28
Gel Drying, Continued
DryEase® Mini-Gel
Drying System,
continued
7. Place one side of the DryEase® Gel Drying Frame with the corner pin facing
up, on the DryEase® Gel Drying Base.
8. Center a piece of pre-wetted cellophane from Step 5 over the base/frame
combination, so the cellophane lays over the inner edge of the frame.
9. Lay the gel on the center of the cellophane sheet making sure no bubbles are
trapped between the gel and the cellophane. Add some Gel-Dry™ Drying
Solution to the surface of the cellophane, if necessary.
10. Carefully lay the second sheet of cellophane over the gel so that no bubbles are
trapped between the cellophane and the gel. Add some Gel-Dry™ Drying
Solution if necessary. Gently smooth out any wrinkles in the assembly with a
gloved hand.
11. Align the remaining frame so that its corner pins fit into the appropriate holes
on the bottom frame. Push the plastic clamps onto the four edges of the
frames.
12. Lift the frame assembly from the DryEase® Gel Drying Base and pour off the
excess solution from the base.
13. Place the gel dryer assembly upright on a benchtop. Be careful to avoid drafts
as they can cause an uneven rate of dying which leads to cracking. Drying will
take between 12–36 hours depending on humidity and gel thickness.
14. When the cellophane is dry to touch, remove the gel/cellophane sandwich
from the drying frame. Trim off the excess cellophane.
15. Press the dried gel(s) between the pages of a notebook under light pressure for
approximately 2 days. Gels will then remain flat for scanning, photography,
display, and overhead projection.
Continued on next page
29
Gel Drying, Continued
Vacuum Drying
General guidelines are provided below to minimize cracking during vacuum
drying of gels. For detailed instructions, follow the manufacturer’s
recommendations.
Handle Gels with Care:
Remove the gel from the cassette without breaking or tearing the edges. Small
nicks or tears can act as a starting point for cracking. Remove the gel wells and
foot off the bottom of the gel with a Gel Knife or a razor blade as described on
page 19. Use the StainEase Staining Tray for staining and destaining gels. This
tray is designed to facilitate the solution changing process without handling of
gels.
Use a Gel Drying Solution:
We recommend equilibrating the gel in a gel drying solution such as Gel-Dry™
Gel Drying Solution for 10-30 minutes at room temperature with gentle shaking
on an orbital shaker before drying the gel. Gel-Dry™ Gel Drying Solution
contains a proprietary non-glycerol component to effectively regulate the rate
of drying and prevent cracking. The gel drying solution does not interfere with
autoradiography.
To prepare your own gel drying solution, prepare a solution containing
30% methanol and 5% glycerol.
Note: Do not incubate coomassie® stained gels in gel drying solution for more
than 5 minutes as the bands may fade.
Remove Air Bubbles:
Remove any air bubbles that may be trapped between the paper, gel, and
plastic wrap by rolling a small glass pipette over the gel. Use additional gel
drying solution to remove any air bubbles.
Use Proper Gel Dryer Set-up:
Place the gel on gel dryer with the plastic wrap facing up. Use a proper
working condition vacuum pump and make sure a tight seal is formed by the
vacuum. Dry gels using gel drying conditions set for polyacrylamide gels
which require increasing the temperature to a set value and holding this
temperature throughout the drying cycle. The recommended conditions for
mini-gels are 80°C for 2 hours.
Ensure Gel is Completely Dry:
The gel will crack if the vacuum seal of the heated gel dryer is broken prior to
complete drying of the gel. To ensure the gel is completely dried before
releasing the vacuum seal, follow these tips :
•
Check the temperature of the gel
The temperature of the dried should be the same as the temperature of the
surrounding gel drying surface. If the temperature of the dried gel is
cooler, then the gel is not completely dried.
•
Check for moisture in the tubing connecting the gel dryer to the vacuum
pump
The gel is not completely dried if there is any residual moisture in the
tubing and additional drying time is required.
30
Western Blotting
Introduction
Instructions are provided below for blotting NuPAGE® Gels using the XCell II™
Blot Module (see page 45 for ordering information). For more information on
the XCell II™ Blot Module, refer to the manual (IM-9051). This manual is
available on our Web site at www.invitrogen.com or contact Technical Service
(see page 51).
See page 36 for Semi-Dry Blotting.
NuPAGE®
Antioxidant
The NuPAGE® Antioxidant is used in the transfer buffer for blotting reduced
proteins and prevents the proteins from reoxidizing and maintains the proteins
in a reduced state (see page 14). Proteins are oxidized during blotting at a slower
rate in the neutral pH environment of NuPAGE® blotting than in a higher pH
blotting system. The major cause of reoxidation during blotting is the oxidizing
effect of the anode electrochemistry.
NuPAGE® Transfer
Buffer
We recommend using the NuPAGE® Transfer Buffer (see page 45 for ordering
information) for western transfer of NuPAGE® Gels as the transfer buffer
maintains the neutral pH environment established during NuPAGE®
electrophoresis.
The NuPAGE® Transfer Buffer protects against modification of the amino acid
side chains and is compatible with N-terminal protein sequencing using Edman
degradation.
Materials Supplied
by the User
•
•
•
•
•
•
•
•
Blotting membranes (see page 45 for ordering information)
Filter paper
Methanol (if using PVDF membranes)
XCell II™ Blot module
NuPAGE® Transfer Buffer
NuPAGE® Antioxidant for reduced samples
MagicMark™ Western Protein Standard (see page 45 for ordering
information)
Deionized water
Continued on next page
31
Western Blotting, Continued
Prepare 1000 ml of 1X NuPAGE® Transfer Buffer using the NuPAGE® Transfer
Preparing
®
NuPAGE Transfer Buffer (20X) as follows:
Reduced Samples
Non-Reduced Samples
Buffer
NuPAGE® Transfer Buffer (20X)
NuPAGE® Antioxidant
Methanol
Deionized Water
Total Volume
50 ml
1 ml
100 ml*
849 ml
1000 ml
50 ml
-100 ml*
850 ml
1000 ml
*NuPAGE® Transfer Buffer with 10% methanol provides optimal transfer of a
single gel in the blot module. If you are transferring two gels in the blot module,
increase the methanol content to 20% to ensure efficient transfer of both gels.
See page 47 for a recipe of the NuPAGE® Transfer Buffer, if you are preparing
your own transfer buffer.
Preparing Blotting
Pads
Use about 700 ml of 1X NuPAGE® Transfer Buffer to soak the pads until
saturated. Remove the air bubbles by squeezing the pads while they are
submerged in buffer. Removing the air bubbles is essential as they can block
the transfer of biomolecules if they are not removed.
Preparing
Transfer
Membrane and
Filter
Cut selected transfer membrane and filter paper to the dimensions of the gel or
use Novex® pre-cut membrane/filter paper sandwiches (see page 45 for
ordering information.
•
PVDF membrane—Pre-wet PVDF membrane for 30 seconds in methanol,
ethanol, or isopropanol. Briefly rinse in deionized water, then place in a
shallow dish with 50 ml of 1X NuPAGE® Transfer Buffer for several
minutes.
•
Nitrocellulose—Place the membrane directly into a shallow dish
containing 50 ml of 1X NuPAGE® Transfer Buffer for several minutes.
•
Filter paper—Soak the filter paper briefly in 1X NuPAGE® Transfer Buffer
immediately prior to use.
•
Gel—Use the gel immediately following the run. Do not soak the gel in
transfer buffer.
Continued on next page
32
Western Blotting, Continued
Western Transfer
Using the XCell II™
Blot Module
Wear gloves while performing the blotting procedure to prevent contamination
of gels and membranes, and exposure to irritants commonly used in
electrotransfer.
Transferring One Gel
1.
After opening the gel cassette as described on page 19, remove wells with
the Gel Knife.
2.
Place a piece of pre-soaked filter paper on top of the gel, and lay just above
the slot in the bottom of the cassette, leaving the “foot” of the gel
uncovered. Keep the filter paper saturated with the transfer buffer and
remove all trapped air bubbles by gently rolling over the surface using a
glass pipette as a roller.
3.
Turn the plate over so the gel and filter paper are facing downwards over a
gloved hand or clean flat surface.
4.
Use the Gel Knife to push the foot out of the slot in the plate and the gel
will fall off.
5.
When the gel is on a flat surface, cut the “foot” off the gel with the gel
knife.
6.
Wet the surface of the gel with transfer buffer and position the pre-soaked
transfer membrane on the gel, ensuring all air bubbles have been removed.
7.
Place another pre-soaked anode filter paper on top of the membrane.
Remove any trapped air bubbles.
8.
Place two soaked blotting pads into the cathode (-) core of the blot module.
The cathode core is the deeper of the two cores and the corresponding
electrode plate is a darker shade of gray. Carefully pick up the gel
membrane assembly and place on blotting pad in the same sequence, such
that the gel is closest to the cathode core (see Figure 1, next page).
9.
Add enough pre-soaked blotting pads to rise to 0.5 cm over rim of cathode
core. Place the anode (+) core on top of the pads. The gel/membrane
assembly should be held securely between the two halves of the blot
module ensuring complete contact of all components.
10. Position the gel/membrane assembly and blotting pads in the cathode core
of the XCell II™ Blot Module to fit horizontally across the bottom of the
unit. There should be a gap of approximately 1 cm at the top of the
electrodes when the pads and assembly are in place.
11. Hold the blot module together firmly and slide it into the guide rails on the
lower buffer chamber. The blot module will only fit into the unit one way,
so the (+) sign can be seen in the upper left hand corner of the blot module.
Properly placed, the inverted gold post on the right hand side of the blot
module will fit into the hole next to the upright gold post on the right side
of the lower buffer chamber.
12. Place the Gel Tension Wedge so that its vertical face is against the blot
module. Lock the Gel Tension Wedge by pulling the lever forward.
Continued on next page
33
Western Blotting, Continued
Western Transfer
Using the XCell II™
Blot Module,
continued
13. Fill the blot module with 1X NuPAGE® Transfer Buffer until the
gel/membrane assembly is covered in this buffer. Do not fill all the way to
the top as this will only generate extra conductivity and heat.
14. Fill the Outer Buffer Chamber with deionized water by pouring
approximately 650 ml in the gap between the front of the blot module and
the front of the lower buffer chamber. The water level should reach
approximately 2 cm from the top of the lower buffer chamber. This serves
to dissipate heat produced during the run.
15. Place the lid on top of the unit.
16. With the power turned off, plug the red and black leads into the power
supply. Refer to Recommended Transfer Conditions on page the next
page for transfer conditions.
Transferring Two Gels in One Blot Module
+
1.
Prepare 1X NuPAGE® Transfer Buffer containing 20% methanol as
described on page 32.
2.
Repeat Steps 1–7 above twice to make two gel/membrane sandwiches.
3.
Place two pre-soaked pads on cathode shell of blot module. Place first
gel/membrane assembly on pads in correct orientation, so gel is closest to
the cathode core. (See Figure 2).
4.
Add another pre-soaked blotting pad on top of first membrane assembly.
5.
Position second gel/membrane assembly on top of blotting pad in the
correct orientation so the gel is closest to the cathode side.
6.
Proceed with steps 8–13 from Transferring One Gel.
7.
Refer to Recommended Transfer Conditions on the next page for transfer
conditions.
Figure 1
Blotting Pad
Blotting Pad
+
Figure 2
Blotting Pad
Blotting Pad
Filter Paper
Filter Paper
Transfer Membrane
Gel
Filter Paper
Transfer Membrane
Second Gel
Filter Paper
Blotting Pad
Blotting Pad
Blotting Pad
Filter Paper
Transfer Membrane
First Gel
Filter Paper
Cathode Core (-)
Blotting Pad
Blotting Pad
Cathode Core (-)
Continued on next page
34
Western Blotting, Continued
Recommended
Transfer
Conditions
Gel
The transfer conditions for NuPAGE® Gels using the XCell II™ Blot Module are
listed in the table below.
Note: The expected current listed in the table is for transferring one gel. If you
are transferring two gels in the blot module, the expected current will double.
Transfer Buffer
®
NuPAGE Novex
Bis-Tris Gel
®
1X NuPAGE Transfer
Buffer with 10% methanol*
Membrane
Power Conditions
Nitrocellulose or
PVDF
30 Volts constant for 1 hour
0.1% NuPAGE® Antioxidant
for reduced samples
®
NuPAGE Novex
Tris-Acetate Gel
®
1X NuPAGE Transfer
Buffer with 10% methanol*
0.1% NuPAGE® Antioxidant
for reduced samples
Expected Current
Start: 170 mA
End: 110 mA
Nitrocellulose or
PVDF
30 Volts constant for 1 hour
Expected Current
Start: 220 mA
End: 180 mA
®
*NuPAGE Transfer Buffer with 10% methanol provides optimal transfer conditions when blotting a single gel in a blot unit. If
transferring two gels within a blot unit, increase the methanol content to 20% to ensure even and efficient transfer of both gels.
Alternate Transfer
Buffers
The NuPAGE® Transfer buffer (with NuPAGE Antioxidant, if samples are
reduced) is the optimal buffer for western transfer of NuPAGE® gels. However,
you can use the Tris-Glycine Transfer Buffer (1X) or TBE Transfer Buffer (1/2X)
for blotting NuPAGE® gels. The NuPAGE® Antioxidant is less functional if
added to the Tris-Glycine and TBE buffers.
Carbonate and CAPS transfer buffers are not recommended for blotting of
NuPAGE® Novex Pre-Cast Gels. The NuPAGE® Antioxidant is ineffective at
pH> 9 and will not work when used with the Carbonate or CAPS transfer
buffers.
Continued on next page
35
Western Blotting, Continued
Semi-Dry Blotting
of NuPAGE®
Novex Bis-Tris
Gels
The NuPAGE® Novex Bis-Tris Gels do not transfer efficiently using a semi-dry
transfer cell as compared to blotting with XCell II™ Blot Module. If you decide
to use semi-dry blotting for NuPAGE® Novex Bis-Tris Gels, use the protocol
provided below to ensure efficient transfer of proteins.
1.
2.
Prepare 100 ml of 2X NuPAGE® Transfer Buffer from 20X NuPAGE®
Transfer Buffer as follows:
NuPAGE® Transfer Buffer (20X)
10.0 ml
®
NuPAGE Antioxidant (for reduced sample) 0.1 ml
Methanol
10.0 ml
Deionized Water
79.9 ml
Total Volume
100 ml
If you are blotting large proteins, see the Note below.
Soak the filter paper and transfer membrane in the transfer buffer.
•
3.
4.
If you are using Novex® pre-cut membrane/filter sandwiches, use three
filter papers (0.4 mm/filter in thickness) on each side of the gel or
membrane.
• If you are not using the Novex® pre-cut membrane/filter sandwiches,
use two thick filter papers.
Assemble the gel/membrane/filter paper sandwich on top of the anode
plate as follows:
Filter Paper
Filter Paper
Filter Paper
Membrane
Gel
Filter Paper
Filter Paper
Filter Paper
Perform the transfer at 15 V constant for 15 minutes if you are using the
Bio-Rad Trans-Blot Semi-Dry Transfer Cell. For any other semi-dry transfer
cell, follow the manufacturer’s recommendations.
Note: For transfer of large proteins (>100 kDa), pre-equilibrate the gel in
2X NuPAGE® Transfer Buffer (without methanol) containing 0.02-0.04%
SDS for 10 minutes before assembling the sandwich.
36
Using ZOOM® Gels
ZOOM® Gels
ZOOM® Gels are 8 x 8 cm, 1.0 mm thick pre-cast polyacrylamide gels cast in a
10 x 10 cm cassette. The ZOOM® Gels are used for 2D analysis of proteins
following isoelectric focusing of 7.0 cm IPG strips. ZOOM® Gels contain an IPG
well and a molecular weight marker well. The IPG well is designed to
accommodate a 7.0 cm IPG strip.
Two types of ZOOM® Gels are available (see page 45 for ordering information)
•
NuPAGE® Novex 4-12% Bis-Tris ZOOM® Gel
•
Novex® 4-20% Tris-Glycine ZOOM® Gel
Second
Dimension
Electrophoresis
The second dimension electrophoresis procedure involves reducing and
alkylating the proteins focused on your IPG strip in equilibration buffer,
loading the strip on your second dimension gel, and performing SDS-PAGE.
Before Starting
You will need the following items:
Equilibrating the
IPG Strip
•
4X NuPAGE® LDS Sample Buffer (see page 45 for ordering information)
•
NuPAGE® Sample Reducing Agent (see page 45 for ordering information)
•
NuPAGE® Novex 4-12% Bis-Tris ZOOM® Gel or Novex® 4-20% TrisGlycine ZOOM™ Gel (see page 45 for ordering information)
•
Appropriate running buffer depending on the type of gel you are using
(see page 45 for ordering information)
•
0.5% agarose solution
•
Iodoacetamide
•
Plastic flexible ruler or thin weighing spatula
•
15 ml conical tubes
•
Water bath set at 55°C or 65°C
•
XCell SureLock™ Mini-Cell (see page 45 for ordering information)
•
Protein molecular weight marker (see page 45 for ordering information)
1.
Dilute 4X NuPAGE® LDS Sample Buffer to 1X with deionized water.
2.
Add 500 µl of the NuPAGE® Sample Reducing Agent to 4.5 ml of the
1X NuPAGE® LDS Sample Buffer from Step 1 in a 15 ml conical tube. Place
one IPG strip in this conical tube for equilibration.
3.
Incubate for 15 minutes at room temperature. Decant the Reducing Solution.
4.
Prepare 125 mM Alkylating Solution fresh by adding 116 mg of
iodoacetamide to 5 ml of 1X NuPAGE® LDS Sample Buffer from Step 1.
5.
Add 5 ml of Alkylating Solution (from Step 4) to the conical tube containing
the IPG strip. Incubate for 15 minutes at room temperature.
6.
Decant the Alkylating Solution and proceed to SDS-PAGE, next page. Use
the equilibrated IPG strip immediately for second dimension.
Continued on next page
37
Using ZOOM® Gels, Continued
SDS-PAGE
A protocol for SDS-PAGE is provided below using ZOOM® Gels with the XCell
SureLock™ Mini-Cell. You may download the XCell SureLock™ Mini-Cell manual
from our web site at www.invitrogen.com or contact Technical Service (see
page 51). If you are using any other electrophoresis system, refer to the
manufacturer’s recommendations.
1.
Prepare 0.5% agarose solution in the appropriate running buffer and keep it
warm (55-65°C) until you are ready to use the agarose solution.
2.
If the molecular weight marker well is bent, straighten the well using a gel
loading tip.
3.
Cut the plastic ends of the IPG strip flush with the gel. Do not cut off any
portions of the gel.
4.
Slide the IPG strip into the ZOOM® Gel well.
5.
Align the IPG strip properly in the ZOOM® Gel well using a thin plastic
ruler or a weighing spatula. Avoid introducing any air bubbles while
sliding the strip.
6.
Pour ~ 400 µl of 0.5% agarose solution into the ZOOM® Gel well containing
the IPG strip. Take care that the agarose solution does not overflow into the
molecular weight marker well.
7.
Assemble the gel cassette/Buffer Core sandwich as described in the XCell
SureLock™ Mini-Cell manual. If you are using only one gel, use the Buffer
Dam to replace the second gel cassette.
Note: Do not use the ZOOM® IPGRunner™ Core for electrophoresis of the
second dimension gel. You must use the Buffer Core supplied with the
XCell SureLock™ Mini-Cell.
8.
Fill the Lower Buffer Chamber and Upper Buffer Chamber with the
appropriate running buffer.
9.
Load molecular weight standards in the marker well.
10. Place the XCell SureLock™ Mini-Cell lid on the Buffer Core. With the power
on the power supply turned off, connect the electrode cords to the power
supply [red to (+) jack, black to (-) jack].
11. Electrophorese at 200 V for 40 minutes for NuPAGE® Novex Bis-Tris
ZOOM® Gel or at 125 V for 90 minutes for Novex® Tris-Glycine ZOOM®
Gel.
12. At the end of electrophoresis, turn off the power and disassemble the gel
cassette/Buffer Core sandwich assembly as described in the XCell
SureLock™ Mini-Cell manual.
13. Proceed to staining the second dimension gel using any method of choice.
38
Calibrating Protein Molecular Weight
Introduction
The molecular weight of a protein can be determined based upon its relative
mobility by constructing a standard curve with protein standards of known
molecular weights.
The protein mobility in SDS-PAGE gels is dependent on the
•
Length of the protein in its fully denatured state,
•
SDS-PAGE buffer systems, and
•
Secondary structure of the protein
The same molecular weight standard may have slightly different mobility,
resulting in different apparent molecular weight when run in different SDSPAGE buffer systems.
If you are using the Novex® protein molecular weight standards, see the
apparent molecular weights of these standards in the NuPAGE® Gels listed on
the next page to determine an apparent molecular weight of your protein.
Protein Secondary
Structure
When using SDS-PAGE for molecular weight determination, slight deviations
from the calculated molecular weight of a protein (calculated from the known
amino acid sequence) can occur due to the retention of varying degrees of
secondary structure in the protein, even in the presence of SDS. This
phenomenon is observed in highly organized secondary structures (collagens,
histones, or highly hydrophobic membrane proteins) and in peptides, where
the effect of local secondary structure becomes magnified relative to the total
size of the peptide.
Buffer Systems
Slight differences in protein mobilities also occur when the same proteins are
run in different SDS-PAGE buffer systems. Each SDS-PAGE buffer system has a
different pH, which affects the charge of a protein and its binding capacity for
SDS. The degree of change in protein mobility is usually small in natural
proteins but more pronounced with “atypical” or chemically modified proteins
such as pre-stained standards.
Continued on next page
39
Calibrating Protein Molecular Weight, Continued
Assigned Apparent
Molecular Weights
The apparent molecular weight values currently provided with the Novex®
molecular weight standards were derived from the construction of a calibration
curve in the Tris-Glycine SDS-PAGE System. We have now calculated and
assigned apparent molecular weights for the Novex® protein standards in several
buffer systems including the NuPAGE® buffer system. Remember to use the one
that matches your gel for the most accurate calibration of your protein.
The following charts summarize the approximate molecular weight values for the
Novex® protein molecular weight standards when run in the NuPAGE® Buffer
System. You may generate calibration curves in your lab with any other
manufacturer’s standards.
Mark 12™ Unstained Standard
NuPAGE® (4-12%)
Bis-Tris/MES
NuPAGE® (4-12%)
Bis-Tris/MOPS
NuPAGE® (3-8%)
Tris-Acetate
200 kDa
200 kDa
200 kDa
β Galactosidase
116.3 kDa
116.3 kDa
116.3 kDa
Phosphorylase B
97.4 kDa
97.4 kDa
97.4 kDa
Bovine Serum Albumin
66.3 kDa
66.3 kDa
66.3 kDa
Glutamic Dehydrogenase
55.4 kDa
55.4 kDa
55.4 kDa
Lactate Dehydrogenase
36.5 kDa
36.5 kDa
36.5 kDa
31 kDa
31 kDa
31 kDa
Trypsin Inhibitor
21.5 kDa
21.5 kDa
N/A
Lysozyme
14.4 kDa
14.4 kDa
N/A
Aprotinin
6 kDa
6 kDa
N/A
Insulin B Chain
3.5 kDa
N/A
N/A
Insulin A Chain
2.5 kDa
N/A
N/A
Myosin
Carbonic Anhydrase
MultiMark® Multi-Colored Standard
NuPAGE® (4-12%)
Bis-Tris/MES
NuPAGE® (4-12%)
Bis-Tris/MOPS
NuPAGE® (3-8%)
Tris-Acetate
Myosin
185 kDa
188 kDa
209 kDa
Phosphorylase B
98 kDa
97 kDa
111 kDa
Glutamic Dehydrogenase
52 kDa
52 kDa
52 kDa
Carbonic Anhydrase
31 kDa
33 kDa
34 kDa
Myoglobin—Blue
19 kDa
21 kDa
N/A
Myoglobin—Red
17 kDa
19 kDa
N/A
Lysozyme
11 kDa
12 kDa
N/A
Aprotinin
6 kDa
N/A
N/A
Insulin
3 kDa
N/A
N/A
Continued on next page
40
Calibrating Protein Molecular Weight, Continued
Assigned Apparent Molecular Weights, continued
SeeBlue® Pre-Stained
Standard
NuPAGE® (4-12%)
Bis-Tris/MES
NuPAGE® (4-12%)
Bis-Tris/MOPS
NuPAGE® (3-8%)
Tris-Acetate
Myosin
188 kDa
191 kDa
210 kDa
BSA
62 kDa
64 kDa
71 kDa
Glutamic Dehydrogenase
49 kDa
51 kDa
55 kDa
Alcohol Dehydrogenase
38 kDa
39 kDa
41 kDa
Carbonic Anhydrase
28 kDa
28 kDa
N/A
Myoglobin
18 kDa
19 kDa
N/A
Lysozyme
14 kDa
14 kDa
N/A
Aprotinin
6 kDa
N/A
N/A
Insulin
3 kDa
N/A
N/A
NuPAGE® (4-12%)
Bis-Tris/MES
NuPAGE® (4-12%)
Bis-Tris/MOPS
NuPAGE® (3-8%)
Tris-Acetate
Myosin
188 kDa
191 kDa
210 kDa
Phosphorylase B
98 kDa
97 kDa
111 kDa
BSA
62 kDa
64 kDa
71 kDa
Glutamic Dehydrogenase
49 kDa
51 kDa
55 kDa
Alcohol Dehydrogenase
38 kDa
39 kDa
41 kDa
Carbonic Anhydrase
28 kDa
28 kDa
N/A
Myoglobin
17 kDa
19 kDa
N/A
Lysozyme
14 kDa
14 kDa
N/A
Aprotinin
6 kDa
N/A
N/A
Insulin
3 kDa
N/A
N/A
SeeBlue® Plus2 Pre-Stained
Standard
41
Troubleshooting
Introduction
Problem
Review the information below to troubleshoot your experiments with
NuPAGE® Gels.
Cause
Solution
Run taking longer
time
Running buffer too dilute
Make fresh running buffer as described on
page 14 and do not adjust the pH of the 1X
running buffer.
Low or no current
during the run
Incomplete circuit
• Remove the tape from the bottom of the
cassette prior to electrophoresis.
• Make sure the buffer covers the sample
wells.
• Check the wire connections on the buffer
core to make sure the connections are intact.
Streaking of proteins
Dumbbell shaped
bands after
electrophoresis
• Sample overload
• Load the appropriate amount of protein as
described on page 6.
• High salt concentration in
the sample
• Decrease the salt concentration of your
sample using dialysis or gel filtration.
• Sample precipitates
• Increase the concentration of SDS in your
sample, if necessary to maintain the
solubility of the protein.
• Contaminants such as
membranes or DNA
complexes in the sample
• Centrifuge or clarify your sample to remove
particulate contaminants.
Loading a large volume of
sample causes incomplete
stacking of the entire sample.
This effect is more intensified
for larger proteins
Load the appropriate volume of sample per
well as described on page 6. If your sample is
too dilute, concentrate the sample using
ultrafiltration.
Continued on next page
42
Troubleshooting, Continued
Using incorrect
Buffers with
NuPAGE® Bis-Tris
Gels
See the table below for the outcome of your results if you accidentally used an
incorrect buffer instead of the NuPAGE® MOPS/MES SDS Running Buffer and
NuPAGE® LDS Sample Buffer on the NuPAGE® Bis-Tris Gels.
If you used the…
NuPAGE® MES SDS
Running Buffer
Instead of the….
NuPAGE® MOPS SDS
Running Buffer
Then….
the run time of the gel is decreased by
~10-15 minutes.
there is decreased separation and
resolution for proteins > 36 kDa.
NuPAGE® MOPS SDS
Running Buffer
NuPAGE® MES SDS
Running Buffer
the run time of the gel is increased by
~10-15 minutes.
the lower molecular weight proteins
(<14 kDa), which are normally well
resolved, are not resolved while the high
molecular weight proteins are resolved
more than normal.
Novex® Tris-Glycine
SDS Sample Buffer
NuPAGE® LDS Sample
Buffer
some bands are not very sharp and there
is increased protein fragmentation.
Novex® Tricine SDS
Sample Buffer
NuPAGE® LDS Sample
Buffer
the band sharpness is not affected, but
the lanes will be slightly wider due to the
increased amount of SDS and buffer salts
from the Tricine Sample Buffer.
Novex® Tris-Glycine
SDS Running Buffer
and the Novex® TrisGlycine SDS Sample
Buffer
NuPAGE® MOPS or
MES SDS Running
Buffer and the
NuPAGE® LDS Sample
Buffer
the gel will have an extremely long run
time of 3-4 hours due to the low
migration of the glycine ions at neutral
pH.
the sensitivity of the staining for high
molecular weight proteins is decreased.
the bands are more compressed at the
bottom of the gel, regardless of the gel
percentage and the bands have a cupped
appearance at the bottom of the band.
Novex® Tricine SDS
Running Buffer and the
Tricine SDS Sample
Buffer
NuPAGE® MOPS or
MES SDS Running
Buffer and the
NuPAGE® LDS Sample
Buffer
the run time of the gel is increased by
1-2 hours due to the slow migration of
the tricine ions at neutral pH.
there may be background streaking in
the lanes.
Continued on next page
43
Troubleshooting, Continued
Using incorrect
Buffers with
NuPAGE® TrisAcetate Gels
Sample Buffer
Running Buffer
Antioxidant
Results
Novex® Tris-Glycine
SDS
NuPAGE® Tris-Acetate SDS
Yes
Fuzzy, smeary bands.
Novex® Tricine SDS
NuPAGE® Tris-Acetate SDS
Yes
Bands are not very sharp.
®
44
Refer to the table below for the outcome of your results if you accidentally used
a incorrect buffer system instead of the NuPAGE® Tris-Acetate SDS Running
Buffer and NuPAGE® LDS Sample Buffer on the NuPAGE® Tris-Acetate Gels.
®
NuPAGE LDS
NuPAGE MES SDS or
NuPAGE® MOPS SDS
Yes
Bands are diffuse and have a
“U” shape. More low
molecular weight proteins
are visible.
NuPAGE® LDS
Novex® Tris-Glycine SDS
No
The run time is twice as long
as the Tris-Acetate Buffer
system. The band resolution
is poor.
NuPAGE® LDS
Novex® Tricine SDS
No
The run time is
10-15 minutes faster than the
Tris-Acetate Buffer system.
Reduced protein bands are
diffuse while non-reduced
large molecular weight
protein bands are smeary.
Novex® Tris-Glycine
SDS
Novex® Tris-Glycine SDS
No
The run time is much longer
than the Tris-Acetate Buffer
system and the bands are
very faint with a streaking
background. Fewer low
molecular weight bands are
resolved.
Novex® Tricine SDS
Novex® Tricine SDS
No
The run time is
10-15 minutes faster than the
Tris-Acetate Buffer system
and reduced protein bands
are not very sharp. The
overall performance is
acceptable.
Accessory Products
Electrophoresis
Reagents
A large variety of electrophoresis reagents and apparatus are available from
Invitrogen for the separation and analysis of proteins. Ordering information is
provided below. For more information, visit our Web site at
www.invitrogen.com or call Technical Service (see page 51).
Product
™
Application
Quantity
Catalog no.
XCell SureLock Mini-Cell
For convenient, leak-free vertical
electrophoresis
1 unit
EI0001
XCell II™ Blot Module
For blotting of mini-gels
1 unit
EI9051
PowerEase 500 Power Supply
Programmable power supply
1 unit
EI8600
DryEase® Mini-Gel Drying
System
Even, crack-free drying of gels using
passive evaporation
1 Kit
NI2387
StainEase® Staining Tray
For even staining and destaining of gels
2/pack
NI2400
For even drying of gels
500 ml
LC4025
For preparing protein samples for
denaturing gel electrophoresis
10 ml
250 ml
NP0007
NuPAGE MOPS SDS Running
Buffer (20X)
Denaturing running buffer for NuPAGE
Bis-Tris Gels
®
500 ml
NP0001
NuPAGE® MES SDS Running
Buffer (20X)
Denaturing running buffer for NuPAGE®
Bis-Tris Gels
500 ml
NP0002
NuPAGE® Tris-Acetate SDS
Running Buffer (20X)
Denaturing running buffer for NuPAGE®
Tris-Acetate Gels
500 ml
LA0041
NuPAGE® Antioxidant
For maintaining reducing conditions
15 ml
during electrophoresis and blotting of the
NuPAGE® Gels
NP0005
NuPAGE® Sample Reducing
Agent (10X)
For preparing reduced protein samples
for NuPAGE® Buffers
250 µl
NP0004
10 ml
NP0009
®
™
Gel-Dry Drying Solution
®
NuPAGE LDS Sample Buffer
(4X)
®
®
®
NP0008
NuPAGE Transfer Buffer (20X)
For Western transfer of NuPAGE Gels
125 ml
NP0006
Novex® Tris-Glycine Native
Running Buffer (10X)
Native running buffer for NuPAGE®
Novex Tris-Acetate Gels
500 ml
LC2672
Novex® Tris-Glycine Native
Sample Buffer (2X)
For preparing protein samples for native
gel electrophoresis
20 ml
LC2673
Nitrocellulose Membrane 0.2
µm
Immunoblotting and amino acid analysis
20 blots
LC2000
Invitrolon™ PVDF membranes
0.45 µm
Immunoblotting, protein sequencing, and 20 blots
amino acid analysis
LC2005
PVDF membranes 0.2 µm
LC2002
Continued on next page
45
Accessory Products, Continued
Protein Standards
and Stains
Ordering information for stains and protein molecular weights is provided
below. For more information, visit our Web site at www.invitrogen.com or
contact Technical Service (see page 51).
Product
™
Application
Quantity
Catalog no.
SimplyBlue Safe-Stain
Fast, sensitive, safe Coomassie G-250
staining of proteins in polyacrylamide gels
1L
LC6060
SilverQuest™ Silver Staining
Kit
Sensitive silver staining of proteins
compatible with mass spectrometry
analysis
1 Kit
LC6070
Colloidal Blue Staining Kit
Sensitive colloidal Coomassie G-250
staining of proteins in polyacrylamide gels
1 Kit
LC6025
SilverXpress® Silver
Staining Kit
High-sensitivity, low background protein
and nucleic acid silver staining
1 Kit
LC6100
Mark 12™ Unstained
Standard
For estimating the apparent protein
molecular weight
1 ml
LC5677
MagicMark™ Western
Standard
For protein molecular weight estimation on
western blots
250 µl
LC5600
SeeBlue® Pre-Stained
Standard
For monitoring the progress of your run
and evaluating transfer efficiency
500 µl
LC5625
SeeBlue® Plus2 Pre-Stained
Standard
For visualizing protein molecular weight
range and evaluating transfer efficiency
500 µl
LC5925
MultiMark® Multi-Colored
Standard
For visualizing protein molecular weight
range and evaluating transfer efficiency
500 µl
LC5725
BenchMark® Protein Ladder
For estimating the apparent protein
molecular weight
2 x 250 µl
10747-012
46
Recipes
NuPAGE® MOPS
SDS Running
Buffer
The NuPAGE® MOPS SDS Running Buffer (20X) is available from Invitrogen (see
page 45)
50 mM MOPS
50 mM Tris base
0.1% SDS
1 mM EDTA
pH 7.7
1.
To prepare 500 ml of 20 X NuPAGE® MOPS SDS Running Buffer, dissolve the
following reagents to 400 ml ultrapure water:
MOPS
Tris Base
NuPAGE® MES
SDS Running
Buffer
104.6 g
60.6 g
SDS
10 g
EDTA
3.0 g
2.
Mix well and adjust the volume to 500 ml with ultrapure water.
3.
Store at +4°C. The buffer is stable for 6 months when stored at +4°C.
4.
For electrophoresis, dilute this buffer to 1X with water (see page 15). The pH of
the 1X solution is 7.7. Do not use acid or base to adjust the pH.
The NuPAGE® MES SDS Running Buffer (20X) is available from Invitrogen (see
page 45)
50 mM MES
50 mM Tris base
0.1% SDS
1 mM EDTA
pH 7.3
1.
To prepare 500 ml of 20 X NuPAGE® MES SDS Running Buffer, dissolve the
following reagents to 400 ml ultrapure water:
MES
97.6 g
Tris Base
60.6 g
SDS
10 g
EDTA
3.0 g
2.
Mix well and adjust the volume to 500 ml with ultrapure water.
3.
Store at +4°C. The buffer is stable for 6 months when stored at +4°C.
4.
For electrophoresis, dilute this buffer to 1X with water (see page 15). The pH of
the 1X solution is 7.3. Do not use acid or base to adjust the pH.
Continued on next page
47
Recipes, Continued
NuPAGE® TrisAcetate SDS
Running Buffer
The NuPAGE® Tris-Acetate SDS Running Buffer (20X) is available from Invitrogen
(see page 45)
50 mM Tricine
50 mM Tris base
0.1% SDS
pH 8.24
1.
To prepare 500 ml of 20 X NuPAGE® Tris-Acetate SDS Running Buffer, dissolve
the following reagents to 400 ml ultrapure water:
Tricine
89.5 g
Tris Base
60.6 g
SDS
NuPAGE® Transfer
Buffer
10 g
2.
Mix well and adjust the volume to 500 ml with ultrapure water.
3.
Store at +4°C. The buffer is stable for 6 months when stored at +4°C.
4.
For electrophoresis, dilute this buffer to 1X with water (see page 15). The pH of
the 1X solution is 8.24. Do not use acid or base to adjust the pH.
The NuPAGE® Transfer Buffer (20X) is available from Invitrogen (see page 45)
25 mM Bicine
25 mM Bis-Tris (free base)
1 mM EDTA
pH 7.2
1.
To prepare 125 ml of 20 X NuPAGE® Transfer Buffer, dissolve the following
reagents to 100 ml ultrapure water:
Bicine
10.2 g
Bis-Tris (free base)
13.1 g
EDTA
0.75 g
2.
Mix well and adjust the volume to 125 ml with ultrapure water.
3.
Store at +4°C. The buffer is stable for 6 months when stored at +4°C.
4.
For western transfer, dilute this buffer to 1X with water (see page 32). The pH
of the 1X solution is 7.2. Do not use acid or base to adjust the pH.
Continued on next page
48
Recipes, Continued
NuPAGE® LDS
Sample Buffer
The NuPAGE® LDS Sample Buffer (4X) is available from Invitrogen (see page 45)
106 mM Tris HCl
141 mM Tris base
2% LDS
10% Glycerol
0.51 mM EDTA
0.22 mM SERVA® Blue G250
0.175 mM Phenol Red
pH 8.5
1.
Tris-Glycine
Native Sample
Buffer
To prepare 10 ml of 4 X NuPAGE® LDS Sample Buffer, dissolve the following
reagents to 8 ml ultrapure water:
Tris HCl
0.666 g
Tris Base
0.682 g
LDS
0.800 g
EDTA
0.006 g
Glycerol
4g
®
SERVA Blue G250 (1% solution)
0.75 ml
Phenol Red (1% solution)
0.25 ml
2.
Mix well and adjust the volume to 10 ml with ultrapure water.
3.
Store at +4°C. The buffer is stable for 6 months when stored at +4°C.
4.
For electrophoresis, prepare your samples in this buffer as described on page 11.
The pH of the 1X solution is 8.5. Do not use acid or base to adjust the pH.
The Tris-Glycine Native Sample Buffer (2X) is available from Invitrogen (see
page 45)
100 mM Tris HCl
10% Glycerol
0.0025% Bromophenol Blue
pH 8.6
1.
To prepare 10 ml of 2 X Tris-Glycine Native Sample Buffer, mix the following
reagents:
4 M Tris HCl
4 ml
10% Glycerol
2 ml
0.1% Bromophenol Blue
0.5 ml
Deionized Water
3.5 ml
2.
Mix well and adjust the pH of the solution is 8.6.
3.
Store at +4°C. The buffer is stable for 6 months when stored at +4°C.
4.
Use this buffer to prepare samples for non-denaturing NuPAGE® Tris-Acetate
gel electrophoresis (see page 11).
Continued on next page
49
Recipes, Continued
Tris-Glycine
Native Running
Buffer
The Tris-Glycine Native Running Buffer (10X) is available from Invitrogen (see
page 45)
25 mM Tris base
192 mM Glycine
pH 8.3
1.
50
To prepare 1000 ml of 10 X Tris-Glycine Native Running Buffer, dissolve the
following reagents in 900 ml deionized water:
Tris Base
29 g
Glycine
144 g
2.
Mix well and adjust the volume to 1000 ml with ultrapure water.
3.
Store at room temperature. The buffer is stable for 6 months when stored at
room temperature.
4.
For native electrophoresis, dilute this buffer to 1X with water (see page 16). The
pH of the 1X solution is 8.3. Do not use acid or base to adjust the pH.
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Continued on next page
51
Technical Service, Continued
Limited Warranty
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52
Purchaser Notification
Limited Use Label
License No. 71:
NuPAGE® Bis-Tris
Gel System
NuPAGE® is the subject of one or more of US Patent Nos. 5,578,180, 5,922,185;
6,096,182; 6,143,154; 6,162,338; EP0753142, and owned by Invitrogen. The
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53
References
Kubo, K. (1995) Effect of Incubation of Solutions of Proteins Containing Dodecyl Sulfate on the Cleavage
of Peptide Bonds by Boiling. Anal Biochem. 225, 351-353.
Moos, M Jr., Nguyen, N. Y., Liu, T.Y. (1988) Reproducible High Yield Sequencing of Proteins
Electrophoretically Separated and Transferred to an Inert Support. J. Biol. Chem. 263, 6005-6008.
Laemmli, U. K., (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage
T4. Nature, 227, 680-685.
©1999-2003 Invitrogen Corporation. All rights reserved.
Coomassie® is a registered trademark of Imperial Chemical Industries PLC.
54
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Invitrogen Corporation
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Email: tech_service@invitrogen.com
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Invitrogen Ltd
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