Novex Midi Gel System - Thermo Fisher Scientific

Novex Midi Gel System - Thermo Fisher Scientific
Novex™ Midi Gel System
USER GUIDE
A system for electrophoresis, blotting, and of midi gels
Publication Number MAN0006268
Revision A.0
For Research Use Only. Not for use in diagnostic procedures.
The information in this guide is subject to change without notice.
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©2017 Thermo Fisher Scientific Inc. All rights reserved.
Contents
Product information ..................................................................................................................................... 4
Product description .................................................................................................................................................................................4
Kit contents and storage .........................................................................................................................................................................6
NuPAGE™ electrophoresis system .......................................................................................................................................................7
Tris-Glycine Midi Gel System................................................................................................................................................................9
Tris-Glycine Plus Midi Gel System ....................................................................................................................................................10
Methods ..................................................................................................................................................... 11
Guidelines for samples and markers .................................................................................................................................................11
Prepare sample for denaturing electrophoresis ..............................................................................................................................12
Prepare samples for non-denaturing electrophoresis ....................................................................................................................13
Guidelines for running buffers............................................................................................................................................................14
Prepare running buffer for denaturing electrophoresis ................................................................................................................15
Prepare running buffer for non-denaturing electrophoresis ........................................................................................................16
Perform electrophoresis ........................................................................................................................................................................16
Visualizing bands in Novex™ Midi Gels ...........................................................................................................................................19
SimplyBlue™ SafeStain protocol..........................................................................................................................................................20
SYPRO™ Ruby Protein Gel Stain protocol ........................................................................................................................................21
Western transfer .....................................................................................................................................................................................22
Semi-dry blotting protocol...................................................................................................................................................................22
iBlot™ 2 Gel Transfer Device protocol................................................................................................................................................24
Appendix A ................................................................................................................................................ 25
Troubleshooting .....................................................................................................................................................................................25
Appendix B................................................................................................................................................. 26
Using Novex™ Midi Gels with the Criterion™ Cell .........................................................................................................................26
Appendix C ................................................................................................................................................ 29
Novex™ Midi Gel specifications ..........................................................................................................................................................29
Appendix D................................................................................................................................................ 30
Accessory products................................................................................................................................................................................30
Appendix E ................................................................................................................................................. 32
Documentation and support................................................................................................................................................................32
Novex™ Midi Gel System User Guide
3
Product information
Product description
Introduction
The Novex™ Midi Gel System is a discontinuous SDS-PAGE, pre-cast polyacrylamide midi gel
system designed to perform higher throughput electrophoresis.
Three types of Novex™ Midi Gel System are available for purchase:
Purpose of the
manual
Applications
•
The NuPAGE™ Midi Gel System is a revolutionary neutral pH, discontinuous SDS-PAGE
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 page 7 for details on the NuPAGE™ Electrophoresis System.
•
The Tris-Glycine Midi Gel System is based on the Laemmli System with minor
modifications for maximum performance in the pre-cast format. The separating and
stacking gels of Novex™ Tris-Glycine Midi Gels have a pH of 8.65, unlike traditional
Laemmli gels that have a stacking gel pH of 6.8 and separating gel pH of 8.8. See page 8 for
details on the Tris-Glycine Electrophoresis System.
•
The Tris-Glycine Plus Midi Gel System is based on the Laemmli System and is modified
to provide maximum performance in the pre-cast format with extended product shelf life.
This manual provides the following information:
•
An overview of the Novex™ Midi Gel System, NuPAGE™ Protein Electrophoresis System,
Tris-Glycine Protein Electrophoresis System, and Tris-Glycine Plus Protein Electrophoresis
System
•
Preparing samples and running buffer
•
Instructions for performing SDS-PAGE using the XCell4 SureLock™ Midi-Cell and Criterion™
Cell (Bio-Rad)
•
Protocol for staining using the SimplyBlue™ Safestain and SYPRO™ Ruby Protein Stain
•
Western blotting protocol using a Semi-Dry blotting apparatus or iBlot™ 2 Dry Blotting
System
•
Troubleshooting
The Novex™ Midi Gels are used to perform higher throughput electrophoresis:
•
For separating proteins under denaturing conditions (NuPAGE™ Bis-Tris, NuPAGE™ TrisAcetate, Novex™ Tris-Glycine, and Novex™ Tris-Glycine Plus Midi Gels)
•
For separating proteins under non-denaturing (native) conditions (NuPAGE™ Tris-Acetate
Novex™ Tris-Glycine, and Novex™ Tris-Glycine Plus Midi Gels)
•
For protein sequencing using Edman sequencing (from gels or PVDF)
Note: Do not use the NuPAGE™ Bis-Tris Midi 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.
4
Novex™ Midi Gel System User Guide
Types of gels
Novex™ Midi Gels are available in different acrylamide concentrations and well formats (see the
following table). All gels are available in 1.0 mm thickness only.
Feature
Gel type
Separating Gel Acrylamide
Concentration
Stacking Gel Acrylamide
Concentration
Well Format
Note
Bis-Tris
Tris-Acetate
Tris-Glycine
Tris-Glycine Plus
8%, 10%, 4–12%
3–8%
8%, 10%, 12%,
4–12%, 4–20%,
8–16%
10%, 12%,
4–12%, 4–20%,
8–16%
4%
3.2%
4%
4%
12+2, 20, and 26
12+2, 20, and 26
12+2, 20, and 26
12+2, 20, and 26
Novex™ Midi Gels do not contain SDS. However, the midi gels are designed for performing
denaturing gel electrophoresis.
Choosing a gel for To obtain the best results for your application, it is important to choose the correct gel
percentage, buffer system, and gel format.
your application
A variety of factors affects the choice of a gel. These include:
Size of the protein being separated
Large proteins resolve well on a low percentage gels while small proteins are best resolved on
high percentage gels. The size of the protein usually dictates the acrylamide percentage. If you
do not know the molecular weight of the protein or are separating a wide molecular weight
range of proteins, choose gradient gels.
Amount of available material
The higher the number of wells, the lower the sample loading volume and vice versa (see page 6
for the recommended loading volumes for the various well formats). Based on the amount of
your starting material available, you can choose from a variety of comb types.
Refer to the gel migration chart on our website at thermofisher.com to choose the right gel for
your application. Choose a gel such that the molecules migrate about 70% of the length of gel
for best resolution (shaded area on the gel migration chart).
Compatibility
The size of a Novex™ Midi Gel is 15 cm × 10.3 cm (gel size is 13 cm × 8.3 cm). We recommend
using the XCell4 SureLock™ Midi-Cell (see page 30 for ordering information) for the
electrophoresis of Novex™ Midi Gels to obtain optimal and consistent performance.
The Novex™ Midi Gels with Midi Gel Adapters are also compatible for use with the Criterion™
Cell available from Bio-Rad.
Downstream
applications
•
The Novex™ Midi Gels are compatible with most staining protocols including silver,
Coomassie, and fluorescent stains.
•
The Novex™ Midi Gels are suited for western transfer applications using a semi-dry or semiwet transfer apparatus that can accommodate a midi gel, or the iBlot™ 2 Gel Transfer Device
(page 22).
Novex™ Midi Gel System User Guide
5
Kit contents and storage
Types of products This manual is intended for use with the following products. For ordering information, visit
thermofisher.com or contact Technical Support (page 32).
Product
Box of 10 gels
NuPAGE™ Novex™ Bis-Tris Midi Gels with Adapters
Box of 10 gels
Box of 10 Midi Gel Adapters
NuPAGE™ Novex™ Tris-Acetate Midi Gels
Box of 10 gels
NuPAGE™ Novex™ Tris-Acetate Midi Gels with Adapters
Box of 10 gels
Box of 10 Midi Gel Adapters
Novex™ Tris-Glycine Midi Gels
Box of 10 gels
Novex™ Tris-Glycine Midi Gels with Adapters
Box of 10 gels
Box of 10 Midi Gel Adapters
Novex™ Tris-Glycine Plus Midi Gels
Box of 10 gels
Novex™ Tris-Glycine Plus Midi Gels with Adapters
Box of 10 gels
Box of 10 Midi Gel Adapters
™
The table below describes the shipping and storage of Novex™ Midi Gels. Do not freeze Novex™
Midi gels.
Shipping and
storage
Item
Shipping
Storage
NuPAGE™ Novex™ Bis-Tris Midi Gels
Room temperature
2°C to 8ºC
12 months
NuPAGE Novex Tris-Acetate Midi Gels
Blue ice
2°C to 8ºC
8 months
Novex™ Tris-Glycine Midi Gels
Blue ice
2°C to 8ºC
4–8 weeks (depending on gel type)
Novex™ Tris-Glycine Plus Midi Gels
Blue ice
2°C to 8ºC
6–12 months (depending on gel type)
Midi Gel Adapters
Room temperature
™
™
Loading volumes
Well types
12 + 2 Well
20 Well
26 Well
6
Quantity
NuPAGE Novex Bis-Tris Midi Gels
™
15°C to 30ºC
Shelf life
Not applicable
The recommended loading volumes and protein load per band by the detection method are
provided in the table below.
Recommended
maximum load
volume
Maximum protein load per band by detection method
Coomassie
staining
45 µL: sample well
15 µL: marker well
0.7 µg/band
25 µL
0.7 µg/band
15 µL
0.4 µg/band
Silver staining
Immunoblotting
Scale your sample load for Scale your sample load
the sensitivity of your silver according to the sensitivity of
staining kit.
your detection method.
A protein load of 1 ng/band
is generally recommended.
Novex™ Midi Gel System User Guide
NuPAGE™ electrophoresis system
System
components
The NuPAGE™ Novex™ Midi Gel System consists of:
•
NuPAGE™ Novex™ Bis-Tris [Bis (2-hydroxyethyl) imino-tris (hydroxymethyl) methaneHCl] Midi Gels for separating small to mid-size molecular weight proteins
•
NuPAGE™ Novex™ Tris-Acetate Midi Gels for separating large molecular weight proteins
•
NuPAGE™ LDS (lithium dodecyl sulfate) Sample Buffer
•
NuPAGE™ Sample Reducing Agent
•
NuPAGE™ Antioxidant
•
NuPAGE™ MES [2-(N-morpholino) ethane sulfonic acid] SDS or MOPS [3-(N-morpholino)
propane sulfonic acid] SDS Running Buffer for NuPAGE™ Novex Bis-Tris Midi Gels
•
NuPAGE™ Tris-Acetate SDS Running Buffer for NuPAGE™ Novex Tris-Acetate Midi Gels
•
NuPAGE™ Transfer Buffer for blotting of NuPAGE™ Novex Midi Gels
NuPAGE™ Novex™ The NuPAGE™ Novex™ Midi Gel is a 1.0 mm thick, wider (13 cm × 8.3 cm) format midi gel used
for higher throughput electrophoresis of protein samples.
Midi Gels
The NuPAGE™ Novex™ Midi Gels are used with the NuPAGE™ Bis-Tris or Tris-Acetate SDS
Buffer System (see page 7) to produce a discontinuous SDS-PAGE system operating at neutral
pH. The neutral pH environment during electrophoresis results in maximum stability of both
proteins and gel matrix, providing better band resolution than other gel systems. See page 5 for
types of gels available from Thermo Fisher Scientific.
NuPAGE™ Bis-Tris The NuPAGE™ Novex™ Bis-Tris discontinuous buffer system involves three ions:
buffer system
• Chloride (Cl–) 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 BisTris (+) and Cl– (pH 6.4).
NuPAGE™ TrisAcetate buffer
system
NuPAGE™ LDS
Sample Buffer
•
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.
Use the NuPAGE™ LDS Sample Buffer (4X) to prepare samples for denaturing gel
electrophoresis with the NuPAGE™ Novex Midi Gels.
The NuPAGE™ LDS Sample Buffer is formulated to reliably provide complete reduction of the
disulfides under mild heating conditions (70ºC for 10 minutes) and eliminate protein cleavage
during sample preparation.
Novex™ Midi Gel System User Guide
7
NuPAGE™ Sample
Reducing Agent
The NuPAGE™ Sample Reducing Agent contains 500 mM dithiothreitol (DTT) at a 10X
concentration and is available in a ready-to-use, stabilized liquid form (page 30). Use the
NuPAGE™ Sample Reducing Agent to prepare samples for reducing gel electrophoresis with
Novex™ Midi Gels.
NuPAGE™
Antioxidant
The reducing agents, DTT and β-mercaptoethanol, do not co-migrate through the gel with the
sample in the neutral pH environment of NuPAGE™ Novex™ Midi Gels. Instead, the reducing
agent tends to remain at the top of the gel and not migrate fully throughout the gel, resulting in
the reoxidization of some proteins, producing 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 of NuPAGE™ gels under
reducing conditions. The NuPAGE™ Antioxidant migrates with the proteins during
electrophoresis of NuPAGE™ gels maintaining sample proteins that have been treated with
reducing agents in a reduced state. The NuPAGE™ Antioxidant also protects sensitive amino
acids such as methionine and tryptophan from oxidizing.
NuPAGE™
Running Buffers
Three NuPAGE™ Running Buffers are available for performing denaturing electrophoresis.
•
NuPAGE™ MES SDS Running Buffer is used with NuPAGE™ Novex™ Bis-Tris Midi Gels to
resolve small molecular weight proteins
•
NuPAGE™ MOPS SDS Running Buffer is used with NuPAGE™ Novex™ Bis-Tris Midi Gels
to resolve mid-size proteins
•
NuPAGE™ Tris-Acetate SDS Running Buffer is used with NuPAGE™ Novex™ Tris-Acetate
Midi Gels to resolve high molecular weight proteins
Note: 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.
NuPAGE™
Transfer Buffer
NuPAGE™ Transfer Buffer is recommended for western transfer of proteins from NuPAGE™
Novex Bis-Tris and Tris-Acetate Midi Gels. The buffer maintains the neutral pH environment
established during gel electrophoresis, protects against modification of the amino acid side
chains, and is compatible with N-terminal protein sequencing using Edman degradation.
Advantages
The operating neutral pH of NuPAGE™ Novex™ Midi Gels and buffers provide the following
advantages over the Laemmli system:
Separation range
•
Longer shelf life of up to 12 months due to improved gel stability
•
Improved protein stability during electrophoresis at neutral pH resulting in sharper band
resolution and accurate results
•
Complete reduction of disulfides with absence of cleavage of Asp-Pro bonds under mild
heating (70°C for 10 minutes) 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™ Novex™ Midi Gels have a wider range of separation throughout the low and
high molecular weight ranges.
By combining any of the NuPAGE™ Novex™ Bis-Tris Midi 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 Midi Gels resolve
proteins in the molecular weight range of 36–400 kDa.
To choose the correct NuPAGE™ Novex™ Midi Gel for your application, refer to the gel
migration chart on our website at thermofisher.com/novex.
8
Novex™ Midi Gel System User Guide
Tris-Glycine Midi Gel System
System
components
Novex™ TrisGlycine Midi Gels
The Novex™ Tris-Glycine Midi Gel System consists of:
•
Novex™ Tris-Glycine Midi Gels for separating a wide range of proteins
•
Novex™ Tris-Glycine SDS Sample Buffer for preparing samples using denaturing conditions
•
Novex™ Tris-Glycine Native Sample Buffer for preparing samples using native conditions
•
NuPAGE™ Sample Reducing Agent (see page 8 for details)
•
Novex™ Tris-Glycine SDS Running Buffer for denaturing electrophoresis
•
Novex™ Tris-Glycine Native Running Buffer for native electrophoresis
•
Novex™ Tris-Glycine Transfer Buffer for blotting of Tris-Glycine Midi Gels
The Novex™ Tris-Glycine Midi Gel is a 1.0 mm thick, wider (13 cm × 8.3 cm) format midi gel
used for higher throughput electrophoresis of protein samples.
The Novex™ Tris-Glycine Midi Gels are used with the Novex™ Tris-Glycine SDS Buffer System
to produce a discontinuous Laemmli SDS-PAGE system. See page 5 for types of gels available
from Thermo Fisher Scientific.
Tris-Glycine
discontinuous
buffer system
The Tris-Glycine discontinuous buffer systems 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 Tris + and Cl–
(pH 8.65).
•
Glycine (–) is the primary anion supplied by the running buffer and serves as a trailing ion.
Glycine is partially negatively charged and trails behind the highly charged chloride ions in
the charged environment. The running buffer ions are Tris +, Gly-, and dodecylsulfate- (pH
8.3).
•
Tris Base (+) is the common ion present in the gel buffer and running buffer. During
electrophoresis, the gel and buffer ions in the Tris-Glycine system form an operating pH of
9.5 in the separation region of the gel.
Novex™ TrisGlycine SDS
Sample Buffer
Use the Novex™ Tris-Glycine SDS Sample Buffer (2X) to prepare samples for denaturing gel
electrophoresis with the Novex™ Tris-Glycine Midi Gels or Novex™ Tris-Glycine Plus Midi Gels.
Novex™ TrisGlycine Transfer
Buffer
Use Tris-Glycine Transfer Buffer for western transfer of proteins from Tris-Glycine or TrisGlycine Plus Midi Gels.
Separation range
The Novex™ Tris-Glycine Midi Gels have a wider range of separation throughout the low and
high molecular weight ranges. The separating range of Tris-Glycine gels is 6–200 kDa.
The Novex™ Tris-Glycine SDS Sample Buffer formulation is based on the Laemmli formulation.
To choose the correct Novex™ Tris-Glycine Midi Gel for your application, refer to the gel
migration chart on our website at thermofisher.com/novex.
Novex™ Midi Gel System User Guide
9
Tris-Glycine Plus Midi Gel System
System
components
The Novex™ Tris-Glycine Plus Midi Gel System consists of:
•
Novex™ Tris-Glycine Plus Midi Gels for separating a wide range of proteins
•
Novex™ Tris-Glycine SDS Sample Buffer for preparing samples using denaturing conditions
(see page 9 for details)
•
Novex™ Tris-Glycine Native Sample Buffer for preparing samples using native conditions
•
NuPAGE™ Sample Reducing Agent (see page 8 for details)
•
Novex™ Tris-Glycine SDS Running Buffer for denaturing electrophoresis
•
Novex™ Tris-Glycine Native Running Buffer for native electrophoresis
•
Novex™ Tris-Glycine Transfer Buffer for blotting of Tris-Glycine Plus Midi Gels (see page 9
for details)
Novex™ TrisGlycine Plus Midi
Gels
The Novex™ Tris-Glycine Plus Midi Gel is a 1.0 mm thick, wider (13 cm × 8.3 cm) format midi
gel used for higher throughput electrophoresis of protein samples.
Tris-Glycine Plus
discontinuous
buffer system
The Tris-Glycine Plus Midi Gels use a proprietary buffer that is based on the Laemmli System
and is modified to provide maximum performance in the pre-cast format with extended
product shelf life.
Separation range
The separating range of Novex™ Tris-Glycine Plus Midi Gels is 6–200 kDa.
The Novex™ Tris-Glycine Plus Midi Gels are used with the Novex™ Tris-Glycine SDS Buffer
System (see page 9) to produce a discontinuous Laemmli SDS-PAGE system. See page 5 for
types of gels available from Thermo Fisher Scientific.
To choose the correct Novex™ Tris-Glycine Midi Gel for your application, refer to the gel
migration chart on our website at thermofisher.com/novex.
10
Novex™ Midi Gel System User Guide
Methods
Guidelines for samples and markers
Reduced and non- For optimal results, it is not recommended to run reduced and non-reduced samples on the
reduced samples same gel.
If it is necessary to run reduced and non-reduced samples on the same gel, follow these
guidelines:
Protein molecular
weight markers
•
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.
•
Do not add NuPAGE™ Antioxidant to the running buffer. The antioxidant will have a
deleterious effect on the non-reduced samples. The bands will appear sharper on NuPAGE™
Midi Gels relative to other gel systems, even without the use of the antioxidant.
The following protein molecular weight markers are recommended for use with the Novex™
Midi Gels (see page 30 for details).
•
PageRuler™ Prestained Protein Ladder
•
PageRuler™ Plus Prestained Protein Ladder
•
NativeMark™ Unstained Protein Ladder
•
HiMark™ Pre-stained and Unstained Protein Standard for determining molecular weight of
large proteins
•
MagicMark™ Western Protein Standard for Western blotting applications
The apparent molecular weight of the protein standards on a midi gel remains approximately
the same as observed for a mini gel.
To obtain equivalent band intensities based on the recommended load for a 10-well mini gel,
use the recommended protein standard load in the following table for each type of midi gel.
Recommended volume for midi gel
Recommended volume for
10-well mini gel
12 + 2 well
20 well
26 well
5 µL
7 µL + 2.5 µL
4 µL
2.5 µL
10 µL
14 µL + 5 µL
8 µL
5 µL
Novex™ Midi Gel System User Guide
11
Prepare sample for denaturing electrophoresis
General
guidelines
Materials needed
Denaturing electrophoresis can be performed using NuPAGE™ Bis-Tris, NuPAGE™ TrisAcetate, Novex™ Tris-Glycine, and Novex™ Tris-Glycine Plus Midi Gels.
•
Add the reducing agent to the sample within an hour before loading the gel.
For best results, add the reducing agent immediately prior to heating.
•
Avoid storing reduced samples for long periods. Even if frozen, samples will reoxidize
during storage and produce inconsistent results.
•
Do not use the NuPAGE™ Antioxidant as a sample reducing agent. The antioxidant is not
efficient in reducing disulfide bonds. Use will result in partially reduced bands with
substantial background smearing in the lane.
You will need the following items. See page 30 for ordering information.
•
Protein sample and molecular weight marker
•
Deionized water
•
Appropriate Sample Buffer
•
Prepare samples
for NuPAGE™ gels
o
NuPAGE™ LDS Sample Buffer (4X) for NuPAGE™ Midi Gels
o
Novex™ Tris-Glycine SDS Sample Buffer (2X) for Tris-Glycine Midi Gels or Novex™
Tris-Glycine Plus Midi Gels
NuPAGE™ Sample Reducing Agent
1. Use the volumes in the following table to prepare samples in a total volume of 10 µL.
To prepare samples in other volumes, scale the volume of reagents accordingly. See page 6
for the recommended protein load.
Reagent
Sample
NuPAGE LDS Sample Buffer (4X)
™
NuPAGE™ Reducing Agent (10X)
Deionized Water
NuPAGE™ Gel
Reduced
Non-Reduced
x µL
x µL
2.5 µL
2.5 µL
1 µL
—
to 10 µL
to 10 µL
2. Heat the sample for denaturing electrophoresis (reduced or non-reduced) at 70°C for
10 minutes for optimal results.
Prepare samples
for Tris-Glycine or
Tris-Glycine Plus
gels
1. Use the volumes in the following table to prepare samples in a total volume of 10 µL.
To prepare samples in other volumes, scale the volume of reagents accordingly. See page 6
for the recommended protein load.
Reagent
Tris-Glycine/ Tris-Glycine Plus Gel
Reduced
Non-Reduced
Sample
x µL
x µL
Tris-Glycine SDS Sample Buffer (2X)
5 µL
5 µL
NuPAGE™ Reducing Agent (10X)
1 µL
—
to 10 µL
to 10 µL
Deionized Water
2. Heat the sample for denaturing electrophoresis (reduced or non-reduced) at 85ºC for
2 minutes for optimal results.
12
Novex™ Midi Gel System User Guide
Prepare samples for non-denaturing electrophoresis
General guidelines
Materials needed
Prepare samples
Non-denaturing (native) electrophoresis can be performed using NuPAGE™ Tris-Acetate,
Novex™ Tris-Glycine, and Novex™ Tris-Glycine Plus Midi Gels.
•
Do not add reducing agent to samples.
•
Do not heat samples for non-denaturing (native) electrophoresis.
You will need the following items. See page 30 for ordering information.
•
Protein sample and molecular weight marker
•
Deionized water
•
Novex™ Tris-Glycine Native Sample Buffer (2X)
1. Use the volumes in the following table to prepare samples in a total volume of 10 µL.
To prepare samples in other volumes, scale the volume of reagents accordingly. See page 6
for the recommended protein load.
Reagent
Volume
Sample
x µL
Novex Tris-Glycine Native Sample Buffer (2X)
5 µL
™
Deionized Water
Novex™ Midi Gel System User Guide
to 10 µL
13
Guidelines for running buffers
General guidelines
Running buffers
Non-denaturing (native) electrophoresis can be performed using NuPAGE™ Tris-Acetate,
Novex™ Tris-Glycine, and Novex™ Tris-Glycine Plus Midi Gels.
•
Do not use the NuPAGE™ Antioxidant with Novex™ Tris-Glycine or Tris-Glycine Plus Midi
Gels.
•
Do not heat samples for non-denaturing (native) electrophoresis.
Five types of running buffers are used for gel electrophoresis of Novex™ Midi Gels. See page 30
for ordering information.
Running buffer
Novex ™ Midi Gels
NuPAGE™
Bis-Tris
NuPAGE™
Tris-Acetate
Tris-Glycine
Tris-Glycine
Plus
Denaturing electrophoresis
NuPAGE MES SDS
Running Buffer (20X)

—
—
—
NuPAGE™ MOPS SDS
Running Buffer (20X)

—
—
—
NuPAGE™ Tris-Acetate SDS
Running Buffer (20X)
—

—
—
Novex™ Tris-Glycine SDS
Running Buffer (10X)
—
—


™
Non-denaturing electrophoresis
Novex™ Tris-Glycine Native
Running Buffer (10X)
Amount of buffer
required for the
XCell4 SureLock™
Midi-Cell
Important
14
—



The amount of 1X Running Buffer required will depend on the number of gels used in the
apparatus as indicated below:
XCell4 SureLock™ Midi-Cell
Number of Gels
Amount of Buffer
4
1400 mL
3
1250 mL
2
950 mL
1
750 mL
In the NuPAGE™ gels, 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.
Novex™ Midi Gel System User Guide
Prepare running buffer for denaturing electrophoresis
Materials needed
•
Deionized water
•
NuPAGE™ Antioxidant for reduced samples with NuPAGE™ Midi Gels
•
Appropriate SDS Running Buffer
•
Prepare
1X NuPAGE™
Running Buffer
o
NuPAGE™ MOPS or MES SDS Running Buffer (20X)
o
NuPAGE™ Tris-Acetate SDS Running Buffer (20X)
o
Novex™ Tris-Glycine SDS Running Buffer (10X)
NuPAGE™ Antioxidant for reduced samples with NuPAGE™ Midi Gels
Use the volumes in the following table to prepare 1000 mL of 1X NuPAGE™ SDS Running
Buffer. Scale-up the volumes accordingly if more buffer is required.
Reducing Conditions
1. Prepare 1000 mL 1X NuPAGE™ SDS Running Buffer using NuPAGE™ SDS Running Buffer
(20X) as follows:
NuPAGE™ SDS Running Buffer (20X)
(MES, MOPS, or Tris-Acetate)
Deionized Water
Total Volume
50 mL
950 mL
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.
3. Immediately, prior to electrophoresis, add NuPAGE™ Antioxidant to 1X NuPAGE™ SDS
Running Buffer from Step 1 for use in the Upper (Inner) Buffer Chamber. Mix thoroughly.
•
For the XCell4 SureLock™ Midi-Cell, add 435 µL NuPAGE™ Antioxidant to 175 mL
1X NuPAGE™ SDS Running Buffer.
•
For the Criterion ™ Cell, add 150 µL NuPAGE™ Antioxidant to 60 mL 1X NuPAGE™ SDS
Running Buffer
Non-Reducing Conditions
1. Prepare 1000 mL 1X NuPAGE™ SDS Running Buffer using NuPAGE™ SDS Running Buffer
(20X) as follows:
NuPAGE™ SDS Running Buffer (20X)
(MES, MOPS, or Tris-Acetate)
Deionized Water
Total Volume
50 mL
950 mL
1000 mL
2. Mix thoroughly and use this buffer in the Lower (Outer) and Upper (Inner) Buffer
Chambers.
Prepare 1X TrisGlycine SDS
Running Buffer
Use the volumes in the following table to prepare 1000 mL of 1X Tris-Glycine SDS Running
Buffer. Scale-up the volume of reagents accordingly if more buffer is required.
1. Prepare 1000 mL 1X Tris-Glycine SDS Running Buffer using Novex™ Tris-Glycine SDS
Running Buffer (10X) as follows:
Tris-Glycine SDS Running Buffer (10X)
Deionized Water
Total Volume
100 mL
900 mL
1000 mL
2. Mix thoroughly and use this buffer in the Lower (Outer) and Upper (Inner) Buffer
Chambers.
Novex™ Midi Gel System User Guide
15
Prepare running buffer for non-denaturing electrophoresis
Materials needed
Prepare 1X Nondenaturing
running nuffer
You will need the following items. See page 30 for ordering information.
•
Deionized water
•
Novex™ Tris-Glycine Native Running Buffer (10X)
Instructions to prepare 1000 mL 1X Tris-Glycine Native Running Buffer are described below.
Scale-up the volume of reagents accordingly if more buffer is needed.
1. Prepare 1000 mL 1X Tris-Glycine Native Running Buffer using Novex™ Tris-Glycine Native
Running Buffer (10X) as follows:
Tris-Glycine Native Running Buffer (10X)
Deionized Water
Total Volume
100 mL
900 mL
1000 mL
2. Mix thoroughly and use this buffer in the Lower (Outer) and Upper (Inner) Buffer
Chambers.
Perform electrophoresis
Introduction
Instructions are provided below for electrophoresis of the Novex™ Midi Gels using the XCell4
SureLock™ Midi-Cell from Thermo Fisher Scientific (page 30).
For electrophoresis of the Novex™ Midi Gels using the Criterion™ Cell from Bio-Rad, see page 26.
Gels are individually packaged in clear pouches with Packaging Buffer. The Packaging Buffer
contains low levels of residual acrylamide monomer and 0.02% sodium azide. Wear gloves 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 SDS, see page 32.
Materials needed
Note
You will need the following items:
•
Appropriate Novex™ Midi Gels (page 6)
•
Protein sample (see page 12 for denaturing electrophoresis, or page 13 for non-denaturing
electrophoresis)
•
1X Running Buffer (page 14)
•
Gel loading tips
•
XCell4 SureLock™ Midi-Cell (page 30)
•
NuPAGE™ Antioxidant for reduced samples for use with NuPAGE™ gels
•
Power Supply (e.g., PowerEase™ 300W Power Supply)
Brief instructions for performing electrophoresis with 4 gels using the XCell4 SureLock™ MidiCell are described on page 17.
For detailed instructions for performing electrophoresis with less than 4 gels, see the XCell4
SureLock™ Midi Cell User Guide supplied with the Midi-Cell or access the manual online at
thermofisher.com
16
Novex™ Midi Gel System User Guide
Electrophoresis
using the XCell4
SureLock™ MidiCell
Instructions for performing electrophoresis with 4 midi gels using the XCell4 SureLock™ MidiCell are described below.
1. Remove the gel cassette from the pouch and rinse with deionized water.
2. Peel off the tape covering the slot on the back of the gel cassette and gently pull the comb
out of the cassette. Rinse the wells with 1X Running Buffer and fill the sample wells with
running buffer.
3. Insert the XCell4 SureLock ™ Assembly in its unlocked position into the center of the MidiCell base.
4. Place one gel cassette on each side of the Buffer Core for each of the cores.
5. While holding the assembly together with your hands, insert the Buffer Cores with gel
cassettes into the Lower Buffer Chamber such that the negative electrode fits into the
opening in the gold plate on the Lower Buffer Chamber. Always hold the assembly by its
edges.
6. Lock the XCell4 SureLock ™ Assembly by moving the tension lever to the locked position
(indicated on the XCell4 SureLock ™ Assembly). This will squeeze the gels and Buffer Cores
together, creating leak free seals.
7. Fill each of the Upper Buffer Chambers with 175 mL of the appropriate 1X Running Buffer.
For reducing conditions with NuPAGE™ Midi Gels, use 1X Running Buffer with 435 µL
NuPAGE™ Antioxidant in each of the Upper Buffer Chambers. Ensure that the Upper Buffer
Chambers are not leaking.
8. Load an appropriate volume of the protein 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 11 for recommended
markers).
10. Add 700 mL 1X Running Buffer (for 4 gels) to the Lower Buffer Chamber (anode) by
pouring into the center of the Midi-Cell (over the XCell4 SureLock™ Assembly). Fill to the fill
line marked on the Midi-Cell.
11. Place the lid on the assembled XCell4 SureLock ™ Midi-Cell. The lid will firmly seat if the (–)
and (+) electrodes are properly aligned.
Run conditions
Perform electrophoresis as described in the table below. Current readings are denoted per gel.
Note: Run times and currents are dependent on gel percentage and power supply.
Midi gel type and buffer system
Voltage
Expected current
Approximate run time
Denaturing electrophoresis
Bis-Tris SDS-PAGE
(MES Running Buffer)
200 V
Start: 160–200 mA
End: 120–170 mA
40 minutes
Bis-Tris SDS-PAGE
(MOPS SDS Running Buffer)
200 V
Start: 160–200 mA
End: 120–170 mA
55 minutes
Tris-Acetate SDS-PAGE
(Tris-Acetate SDS Running Buffer)
150 V
Start: 70–90 mA
End: 50–60 mA
70 minutes
Tris-Glycine Plus
(Tris-Glycine SDS Running Buffer)
200 V
Start: 70–75 mA
End: 35–40 mA
55–65 minutes
Tris-Glycine
(Tris-Glycine SDS Running Buffer)
125 V
Start: 40–50 mA
End: 20–25 mA
105 minutes
Novex™ Midi Gel System User Guide
17
Midi gel type and buffer system
Voltage
Expected current
Approximate run time
Non-denaturing electrophoresis
Tris-Acetate
(Tris-Glycine Native Running Buffer)
150 V
Start: 40–45 mA
End: 15–20 mA
2–3 hours
Tris-Glycine Plus with Tris-Glycine Native
Running Buffer
125 V
Start: 35–40 mA
End: 15–20 mA
105–125 minutes
Tris-Glycine
(Tris-Glycine Native Running Buffer)
125 V
Start: 35–40 mA
End: 15–20 mA
115–125 minutes
Remove gel from
cassette
1. After electrophoresis is complete, shut off the power, disconnect electrodes, and remove the
lid.
2. Unlock the XCell4 SureLock™ Assembly by moving the tension lever to the unlocked
position (indicated on the XCell4 SureLock™ Assembly).
3. Remove the Buffer Cores with the gel cassettes from the Lower Buffer Chamber while
holding the cassettes against the cores. Remove the gel cassettes from the Buffer Cores and
lay the gel cassettes on a flat surface, such as the bench top. The notched (“well”) side of the
cassette should face up.
4. Separate each of the three bonded sides of the cassette by inserting the Gel Knife into the
gap between the cassette’s two plates. 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.
5. Carefully remove and discard the top plate, allowing the gel to remain on the bottom
(slotted) plate.
Note: The Cassette Post (small plastic piece near the top of the cassette) may remain on
either plate of the cassette after opening the two plates of the cassette. The Cassette Post is
designed to maintain proper electrophoresis conditions that result in optimal separation
and does not interfere with the sample loading or the electrophoresis run.
6. If blotting, remove the gel foot and well sections of the gel. Proceed immediately to western
transfer (see page 22).
7. If staining, remove the gel from the plate by one of the following methods:
•
Use the sharp edge of the gel knife to remove the bottom foot 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
foot. 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
foot off of the gel after fixing or staining, but before gel drying.
8. Fix and stain the gel as described on page 19.
18
Novex™ Midi Gel System User Guide
Visualizing bands in Novex™ Midi Gels
Compatible stains
Novex™ Midi Gels are compatible with most staining protocols including silver, Coomassie, and
fluorescent stains. See page 30 for ordering details.
Silver staining
The SilverQuest™ Silver Staining Kit or the SilverXpress™ Silver Staining Kit are recommended
for silver staining of Novex™ Midi Gels.
Coomassie staining
Novex™ Midi Gels are compatible with any of the standard Coomassie staining procedures.
Protocols that are accelerated by heat are preferable as the heat serves to fix proteins, especially
smaller peptides.
SimplyBlue™ SafeStain (see page 20 for a brief protocol) and Novex™ Colloidal Coomassie Blue
Staining Kit are recommended for staining Novex™ Midi Gels.
Fluorescent staining
Novex™ Midi Gels are compatible with fluorescent stains such as the SYPRO™ Ruby Protein Gel
Stain (see page 21 for a brief protocol).
General staining
guidelines
Preservation of
midi gels
You may use any staining protocol of choice. Follow the general guidelines listed below to
obtain the best results:
•
For converting a mini-gel staining protocol to stain the Novex™ Midi Gel, use ~1.5 times the
volume of reagents recommended for a mini-gel.
•
The volume of fixing, staining, and destaining solutions will depend on the volume of your
staining container. To obtain good results, the solution volume must be sufficient to cover
the gel completely and to allow the gel to move freely during all of the steps.
•
When using a microwave oven for staining, be sure the gel is completely covered in the
solution and use a microwaveable staining container. Use caution while using staining
reagents in a microwave oven. Do not overheat the staining solutions.
The stained Midi Gels can be dried for storage or analysis by vacuum-drying or air-drying. We
recommend using the Large Gel Drying Kit (page 30) to air-dry the gel.
Novex™ Midi Gel System User Guide
19
SimplyBlue™ SafeStain protocol
Materials needed
SimplyBlue ™
SafeStain
microwave
protocol
•
Incubation Trays (page 30) or appropriate staining containers
•
Shaker
•
Deionized water
•
SimplyBlue SafeStain
•
Microwave oven
•
20% NaCl (w/v) in deionized water
SimplyBlue SafeStain (page 30) is a ready-to-use, proprietary Coomassie G-250 stain that is
specially formulated for fast, sensitive detection and safe, non-hazardous disposal.
Instructions for staining gels with SimplyBlue™ SafeStain using a microwave oven are included
in this section. For more details, refer to the SimplyBlue SafeStain manual available at
thermofisher.com or contact Technical Support (page 32).
1. After electrophoresis, place the gel in 150 mL ultrapure water in a loosely covered
microwaveable container and microwave on High (950–1100 watts) for 1 minute until the
solution almost boils.
Note: Do not use the Incubation Tray available from Thermo Fisher Scientific as the
Incubation Tray is not heat-resistant and cannot be heated in a microwave oven or
autoclaved.
2. Shake the gel on an orbital shaker for 1 minute. Discard the water.
3. Repeat Steps 1–2 two more times.
4. Add 40 mL SimplyBlue™ SafeStain and microwave on High for 45 seconds to 1 minute until
the solution almost boils.
5. Shake the gel on an orbital shaker for 5 minutes. Discard the stain.
6. Wash the gel in 150 mL ultrapure water for 10 minutes on a shaker.
7. Add 30 mL 20% NaCl to the water in Step 6 and incubate for at least 5 minutes. The gel can
be stored for several weeks in the salt solution.
8. Optional: Repeat Step 6 for 1 hour for a clear background.
20
Novex™ Midi Gel System User Guide
SYPRO™ Ruby Protein Gel Stain protocol
Materials needed
SYPRO™ Ruby
staining protocol
•
Incubation Trays (page 30) or appropriate staining containers
•
Shaker
•
Deionized water
•
SYPRO™ Ruby Protein
•
Fixing solution (20% acetic acid)
•
Destaining solution (10% methanol, 7% acetic acid)
•
UV transilluminator equipped with a standard camera or an appropriate laser scanner
(page 21)
SYPRO™ Ruby Protein Gel Stain (page 30) is a ready-to-use, highly sensitive fluorescent stain for
protein staining.
Instructions for staining gels with SYPRO™ Ruby Protein Gel Stain are included in this section.
For more details, refer to the SYPRO™ Ruby Protein Gel Stain manual available at
thermofisher.com or contact Technical Support (page 32).
1. After electrophoresis, remove the gel from the cassette (page 18) and place the gel in a clean
Incubation Tray.
2. Fix the gel in Staining Solution (20% acetic acid) for 30 minutes on an orbital shaker.
3. Stain the gel in undiluted SYPRO™ Ruby Protein Gel Stain for 1.5 hours on an orbital
shaker.
4. Transfer the gel to a clean Incubation Tray and destain in Destaining Solution (10%
methanol, 7% acetic acid) for ~2 hours. If complete removal of background is desired,
perform the destaining step overnight.
5. Place the gel on a UV transilluminator equipped with a standard camera and select the
ethidium bromide filter on the camera.
You can also use a laser-based scanner with a laser line that falls within the excitation
maxima of the stain (610 nm).
6. Image the gel with a suitable camera with the appropriate filters using a 1-4 second
exposure. You may need to adjust the brightness and contrast to reduce any faint
non-specific bands.
You should see fluorescent protein bands and the gel should have minimal background.
Novex™ Midi Gel System User Guide
21
Western transfer
Compatible
transfer systems
Novex™ Midi Gels can be blotted using any semi-dry or semi-wet transfer apparatus that can
accommodate a a Novex™ Midi Gel (13 cm × 8.3 cm). A semi-dry blotting procedure for blotting
Novex™ Midi Gels is described in the following section.
The gels are also compatible with the iBlot™ 2 Gel Transfer Device using a regular sized transfer
stack (see page 24 for a brief protocol).
General
guidelines
•
Wear gloves at all times during the entire blotting procedure to prevent contamination of
gels and membranes, and to avoid exposing your skin to irritants commonly used in
electrophoresis and blotting procedures.
•
Do not touch the membrane or gel with bare hands. This may contaminate the gel or
membrane and interfere with further analysis.
Semi-dry blotting protocol
Materials needed
Types of transfer
buffer
For ordering information, see page 30.
•
Semi-dry transfer apparatus
•
Methanol
•
NuPAGE™ Transfer Buffer (20X) or Tris-Glycine Transfer Buffer (25X)
•
NuPAGE™ Antioxidant (for use with NuPAGE™ gels)
•
Blotting membranes: Invitrolon™/Filter Paper Sandwich or Nitrocellulose/Filter Paper
Sandwiches
•
4 pieces of 2.5 mm thick Blotting Filter Paper per gel
•
Blotting Roller
•
Incubation Tray
Use appropriate transfer buffer based on the type of midi gel you are using.
NuPAGE™
Bis-Tris
NuPAGE™
Tris-Acetate
Tris-Glycine [1]
Tris-Glycine
Plus [1]
NuPAGE™ Transfer Buffer


—
—
Tris-Glycine Transfer Buffer
—
—


[1] Because Edman protein sequencing is inhibited by Tris, use the NuPAGE™ Transfer Buffer
instead of Tris-Glycine Transfer Buffer if you plan to perform protein sequencing from a
membrane.
Prepare 2X
NuPAGE™
Transfer Buffer
Prepare 500 mL of 2X NuPAGE™ Transfer Buffer with 10% methanol using the NuPAGE™
Transfer Buffer (20X) as follows:
NuPAGE™ Transfer Buffer (20X)
50 mL
0.5 mL
NuPAGE™ Antioxidant (for reduced samples only)
Methanol
50 mL
Deionized Water
to 500 mL
Prepare 2X TrisGlycine Transfer
Buffer
Prepare 500 mL of 2X Tris-Glycine Transfer Buffer with 10% methanol using the Tris-Glycine
Transfer Buffer (25X) as follows:
Tris-Glycine Transfer Buffer (25X)
40 mL
Methanol
50 mL
Deionized Water
to 500 mL
22
Novex™ Midi Gel System User Guide
Equilibrate the gel
Equilibration of the gel in transfer buffer results in the removal of salts that may increase
conductivity and heat during transfer. Perform equilibration for the recommended time, as
longer equilibration can result in protein diffusion.
1. After electrophoresis, remove the gel from the cassette as described on page 18.
2. Equilibrate the Midi Gel in 100 mL of the appropriate 2X Transfer Buffer (see above for
recipes) for 10 minutes on an orbital shaker.
Prepare blotting
membrane
Nitrocellulose
1. Use pre-cut Nitrocellulose/Filter Paper Sandwich or cut nitrocellulose membrane to the
appropriate size (13 cm × 8.3 cm).
2. Soak the membrane in a 2X Transfer Buffer (see above for recipe) for several minutes in the
Incubation Tray.
PVDF
1. Use pre-cut Invitrolon ™/Filter Paper Sandwich or cut PVDF membrane to the appropriate
size (13 cm × 8.3 cm).
2. Pre-wet the membrane for 30 seconds in methanol, ethanol, or isopropanol. Briefly rinse the
membrane in deionized water.
3. Soak the membrane in a 2X Transfer Buffer (see above for recipe) for several minutes in the
Incubation Tray.
Transfer protocol
Instructions are provided below for blotting Tris-Glycine and Tris-Glycine Plus Midi Gels using
a semi-dry blotting apparatus with a lower anode plate.
1. In a clean container or Incubation Tray, briefly soak 2 pieces of 2.5 mm thick Blotting Filter
Paper (8.6 cm × 13.5 cm) in the appropriate 2X Transfer Buffer (see page 22). Several pieces
of thinner blotting paper can be used to produce a stack of equivalent thickness.
2. Remove any air bubbles trapped between filter paper sheets using the Blotting Roller while
the paper is still submerged in buffer.
3. Place the 2 pieces of pre-soaked 2.5 mm thick Blotting Filter Paper from Step 1 (or
equivalent thickness of thinner filter paper) on the anode plate of a semi-dry blotting
apparatus. Remove any air bubbles between the paper and plate with the Blotting Roller.
4. Place the pre-soaked blotting membrane on top of the filter paper stack and remove any air
bubbles with the Blotting Roller.
5. Place the gel on top of the blotting membrane and remove any air bubbles with the Blotting
Roller or a wet gloved finger.
6. Briefly soak 2 additional pieces of 2.5 mm thick Blotting Filter Paper in the appropriate 2X
Transfer Buffer as was done in Step 1, and then gently place them on top of the gel.
7. Ensure that the filter paper sheets are aligned properly and flush with the gel/membrane
sandwich. Remove any air bubbles with the Blotting Roller
8. Place the cathode plate on the stack without disturbing the blot sandwich. Follow the
manufacturer’s instructions to further assemble the semi-dry blotting apparatus.
9. Transfer at 20 V for 1 hour (~33 V/cm). You may need to optimize the transfer conditions
for your specific proteins or semi-dry transfer apparatus.
Novex™ Midi Gel System User Guide
23
iBlot™ 2 Gel Transfer Device protocol
Introduction
Brief instructions are provided below for blotting Novex™ Midi Gels using the iBlot™ 2 blotting
apparatus. For detailed instructions for performing blotting, see the iBlot™ 2 Dry Blotting
System User Guide or access the manual online at thermofisher.com
Equilibrate the gel
Equilibrating the gel in 100 mL of deionized water or transfer buffer for 5 minutes prior to
transfer may improve transfer of mid to small molecular weight proteins (See step 7 in the
following protocol).
This equilibration step can be omitted for optimal transfer of higher molecular weight proteins
(>150kDa).
Transfer protocol
1. Select Method P0 on the iBlot™ 2 Gel Transfer Device.
2. Unseal an iBlot™ 2 Regular Size Transfer Stack.
3. Separate the Top Stack and set it to one side of the bench with the transfer gel layer facing
up. Keep the Bottom Stack in the transparent plastic tray.
4. Place the Bottom Stack with the plastic tray directly on the blotting surface and align the
electrical contacts on the tray with the corresponding electrical contacts on the blotting
surface of the iBlot™ 2 Gel Transfer Device.
Orientation for iBlot® 2 Regular SizeTransfer Stack
Electrical contacts
5. Remove any air bubbles trapped between the membrane and transfer stack with the
Blotting Roller.
6. Remove the gel from the cassette as described on page 18, and cut off the foot of the gel.
7. Immerse the gel in 100 mL of deionized water briefly (1–10 seconds) to facilitate easy
positioning of the gel on top of the transfer membrane.
Note: For Tris-Glycine Plus gels, rinse in 100 mL of deionized water for 5 minutes to
improve transfer of mid to low molecular weight proteins (< 150 kDa).
8. Remove any air bubbles between the gel and membrane with the Blotting Roller.
9. Place a wet iBlot™ Filter Paper on top of the gel and remove any air bubbles with the
Blotting Roller.
10. Place the Top Stack and palce it on top of the filter paper with the copper electrode facing
up. Remove any air bubbles with the Blotting Roller.
11. Place the iBlot™ 2 Absorbent Pad on top of the transfer stack. Ensure the electrical contacts
are aligned with the corresponding contacts on the blotting surface of the iBlot™ 2 Gel
Transfer Device.
12. Flatten any protrusions on the surface of the stack with the Blotting Roller.
13. Close the lid of the iBlot™ 2 Gel Transfer Device gently.
14. Transfer using Method P0 on the iBlot™ 2 Gel Transfer Device.
Note: Blotting parameters (volts or time) may need optimization based on your initial results.
24
Novex™ Midi Gel System User Guide
Appendix A
Troubleshooting
Review the information below to troubleshoot your experiments with Novex™ Midi Gels.
Introduction
Observation
Run taking longer
time
Cause
Running buffer too dilute
Solution
•
•
Low or no current
during the run
Incomplete circuit
•
•
•
•
Streaking of
proteins
•
Sample overload
•
•
High salt concentration in the
sample
Sample precipitates
•
Contaminants such as
membranes or DNA complexes in
the sample
•
•
•
•
Ensure the running buffer corresponds to the type of
gel being used (page 14).
Make fresh running buffer as described on page 15
and do not adjust the pH of the 1X running buffer.
Remove the tape from the bottom of the gel 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.
Ensure the lid is properly positioned and seated
correctly.
Load the appropriate amount of protein as described
on page 6.
Decrease the salt concentration of your sample using
dialysis or gel filtration.
Increase the concentration of SDS in your sample, if
necessary to maintain the solubility of the protein.
Centrifuge or clarify your sample to remove
particulate contaminants.
Dumbbell shaped
bands after
electrophoresis
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.
Poor resolution,
bands are not very
sharp (fuzzy,
smeary, streaking)
Incorrect sample or running buffer
used
Use the recommended sample buffer and 1X Running
Buffer based on the gel type. Do not use the NuPAGE™
Bis-Tris Midi Gels with NuPAGE™ MOPS or MES Running
Buffer without SDS for native gel electrophoresis.
Do not use NuPAGE™ SDS Running Buffer for
electrophoresis of Tris-Glycine or Tris-Glycine Plus Midi
Gels and do not use Tris-Glycine SDS Running Buffer for
electrophoresis of NuPAGE™ Midi Gels.
High background
staining in upper
portion of gel
stained with Simply
Blue SafeStain™
Higher staining of low % acrylamide
portion of the gel
Add 5 mL 20% NaCl (w/v in deionized water) to the gel
rinse after the initial water destain (1 hour at room
temperature if not using the microwave protocol).
Continue to destain for at least 5 minutes, or until the
desired low background is produced.
Novex™ Midi Gel System User Guide
25
Appendix B
Using Novex™ Midi Gels with the Criterion™ Cell
Introduction
To efficiently use the Novex™ Midi Gels with the Criterion ™ Cell from Bio-Rad, you will need to
use the Midi Gel Adapter with the Novex™ Midi Gel. The Midi Gel Adapter is supplied with the
Novex™ Midi Gels with Adapters as well as available separately.
Brief instructions for using the Novex™ Midi Cassette with a Midi Gel Adapter for use with the
Criterion ™ Cell are described in this section. For details on using the Criterion™ Cell, refer to the
manual supplied with the apparatus.
To use a Novex™ Midi Gel with the XCell4 SureLock™ Midi-cell, see page 17.
Note: During electrophoresis, it is normal for electrical current values to be slightly higher and
run times to be slightly shorter for a given run voltage than when the XCell4 Surelock Midi-Cell
is used.
Midi Gel Adapter
Each Midi Gel Adapter is designed with two alignment tabs (indicated with circles in the figure
below) that fit into the slots on the Novex™ Midi Cassette and facilitate the attachment of the
adapter onto the Novex™ Midi Cassette. The Midi Gel Adapter contains an adhesive on the
inner side. After removing the adhesive liner from the adapter and placing the adapter on a dry
surface of the Novex™ Midi Cassette, the adhesive creates a tight seal between the adapter and
cassette and holds each adapter on to the cassette.
Adhesive
The Novex™ Midi Cassette/Adapter assembly makes the Novex™ Midi Gel compatible for use
with the Criterion ™ Cell and creates an upper buffer chamber that can hold ~75 mL of running
buffer for electrophoresis.
Note
Materials needed
26
•
The Midi Gel Adapter is designed for use with the Novex™ Midi Gel in the Criterion ™ Cell
(Bio-Rad) only. Do not use the Midi Gel Adapter with any other electrophoresis apparatus.
•
Do not re-use the Midi Gel Adapter. Discard the adapter after use.
You will need the following items. For ordering information, see page 30.
•
Novex™ Midi Gels with Adapters
•
Protein sample prepared in the appropriate sample buffer (see page 12 for denaturing
electrophoresis, or page 13 for non-denaturing electrophoresis)
•
Appropriate 1X Running Buffer (see page 14)
•
Gel loading tips
•
Criterion ™ Cell (available from Bio-Rad)
•
NuPAGE™ Antioxidant for reduced samples for use with NuPAGE™ gels
•
Power Supply (e.g., PowerEase™ 300W Power Supply)
Novex™ Midi Gel System User Guide
Important
•
To obtain a tight seal, be sure to insert the alignment tabs of the adapter into the two slots of
the cassette and press the adapter firmly on the cassette.
•
Leaks are generally caused:
o
o
•
Attach the midi gel
adapter
When the adapter is not firmly pressed onto the cassette or the surface of the cassette
was wet when the adapter was applied. To obtain a tight seal, press the adapter firmly
on the cassette.
By adding excess buffer. The Midi Gel Adapter is designed to hold ~75 mL buffer.
When leaks occur, it is best to remove the adapter and discard it. Remove any remaining
adhesive on the cassette, dry the cassette with a paper towel and then place a fresh new
adapter on the dried surface of the cassette as described in the following section.
1. Remove one Midi Gel Adapter and one Novex™ Midi Gel from their individual packages.
2. After ensuring that your hands are dry, blot any excess liquid from the cassette using a
paper towel.
3. Locate the slots on the Novex™ Midi Cassette as shown in figure A below. Avoid introducing
any liquid onto the cassette surface.
4. Peel off the Adhesive Liner from the Midi Gel Adapter with dry hands.
5. Hold the Midi Gel Adapter such that the Invitrogen logo is facing towards you (adhesive
side towards the cassette) and align the alignment tabs of the adapter with the two slots on
the cassette. Place the adapter on the cassette and apply firm pressure to the adapter on the
adhesive area to ensure a tight seal between the adapter and cassette (figure B).
Figure A (Cassette only)
Figure B (Cassette with Adapter)
Slot
Slot
The attachment of the adapter on the gel cassette generates an upper buffer chamber that
can hold ~75 mL running buffer and is required for use with the Criterion ™ Cell.
6. Remove the comb from the cassette and rinse the wells with 1X Running Buffer.
7. Remove the tape from the bottom of the cassette.
8. Use the Novex™ Midi Cassette/Adapter assembly immediately for electrophoresis as
described on page 28. We recommend using the cassette/adapter assembly within 1 hour of
assembly to obtain the best results and prevent any leaks.
Amount of buffer
The amount of 1X Running Buffer required will depend on the number of gels used in the
apparatus as indicated below:
For Criterion™ Cell (Bio-Rad)
Number of gels
Novex™ Midi Gel System User Guide
Amount of buffer
2
1000 mL
1
500 mL
27
Perform
electrophoresis
1. Insert the Novex™ Midi Cassette/Adapter assembly into one of the slots in the Criterion ™
Cell tank such that the adapter is facing the center of the cell.
2. Add 60 mL of the appropriate 1X Running Buffer into the upper buffer chamber.
For reduced samples being run on NuPAGE™ gels, use 60 mL 1X Running Buffer containing
150 µL NuPAGE™ Antioxidant. If you notice any leaks, see page 26.
3. Load the appropriate volume of samples and protein molecular weight markers in the wells.
4. Load each half of the lower buffer chamber with 400 mL 1X Running Buffer (for
electrophoresis of 2 gels).
5. Place the lid on the Criterion ™ Cell.
6. With the power off, connect the electrode cords to the power supply. Turn on the power
supply and perform electrophoresis using the following settings:
Midi gel type and buffer system
Voltage
Expected current
Approximate run time
NuPAGE™ Novex™ Bis-Tris with MES SDS
Running Buffer (denaturing, reducing)
200 V
Start: 250–270 mA
End: 150–170 mA
35 minutes
NuPAGE™ Novex™ Bis-Tris with MOPS SDS
Running Buffer (denaturing, reducing)
200 V
Start: 250–270 mA
End: 150–170 mA
40 minutes
NuPAGE™ Novex™ Tris-Acetate with TrisAcetate SDS Running Buffer (denaturing,
reducing)
150 V
Start: 80–100 mA
End: 50–60 mA
60 minutes
NuPAGE™ Novex™ Tris-Acetate with TrisGlycine Native Running Buffer (nondenaturing)
150 V
Start: 50–60 mA
End: 15–20 mA
1.5–2 hours
Tris-Glycine with Tris-Glycine SDS Running
Buffer
125 V
Start: 55–70 mA
End: 20–30 mA
90 minutes (depending on gel type)
Tris-Glycine with Tris-Glycine Native
Running Buffer (non-denaturing)
125 V
Start: 45–55 mA
End: 15–20 mA
100 minutes (depending on gel type)
Tris-Glycine Plus with Tris-Glycine SDS
Running Buffer
200 V
Start: 95–105 mA
End: 35–50 mA
45–55 minutes
Tris-Glycine Plus with Tris-Glycine Native
Running Buffer (native electrophoresis)
125 V
Start: 50–60 mA
End: 15–20 mA
90–105 minutes
Note: Run times and currents are dependent on gel percentage and power supply
7. Disassemble the Criterion ™ Cell as described in the manual supplied with the apparatus.
28
Novex™ Midi Gel System User Guide
Appendix C
Novex™ Midi Gel specifications
Specifications
Gel Matrix:
Acrylamide/Bisacrylamide
Gel Size:
13 cm × 8.3 cm
Gel Thickness:
1.0 mm
Cassette Size:
15 cm × 10.3 cm
Cassette Material:
Styrene Copolymer
Gel Types:
NuPAGE™ Bis-Tris, NuPAGE™ Tris-Acetate,
Novex™ Tris-Glycine, Novex™ Tris-Glycine
Plus
Sample Well Configuration:
12+2, 20, and 26 well
Novex™ Midi Gel System User Guide
29
Appendix D
Accessory products
Ordering information for electrophoresis products available separately from Thermo Fisher
Scientific is provided below. For detailed information, visit our website at thermofisher.com or
call Technical Support (page 32).
Additional
products
Product
Quantity
Catalog No.
1 unit
WR0100
1 kit
LA0050
NuPAGE Tris-Acetate SDS Running Buffer (20X)
500 mL
LA0041
NuPAGE Sample Reducing Agent (10X)
250 µL
NP0004
NuPAGE™ Antioxidant
15 mL
NP0005
NuPAGE™ LDS Sample Buffer (4X)
10 mL
NP0007
NuPAGE Transfer Buffer (20X)
1L
NP0006-1
NuPAGE™ MOPS SDS Buffer Kit
1 kit
NP0050
NuPAGE™ MES SDS Buffer Kit
1 kit
NP0060
NuPAGE MOPS SDS Running Buffer (20X)
500 mL
NP0001
NuPAGE™ MES SDS Running Buffer (20X)
500 mL
NP0002
Novex™ Tris-Glycine SDS Sample Buffer (2X)
20 mL
LC2676
Novex Tris-Glycine SDS Running Buffer (10X)
500 mL
LC2675
Novex™ Tris-Glycine Transfer Buffer (25X)
500 mL
LC3675
Novex™ Tris-Glycine Native Running Buffer (10X)
500 mL
LC2672
Novex Tris-Glycine Native Sample Buffer (2X)
20 mL
LC2673
XCell4 SureLock Midi-Cell

NuPAGE™ Tris-Acetate SDS Buffer Kit
™
™
™
™
™
™
Quantity
Catalog No.

Stains
1L
LC6060

SilverQuest Silver Staining Kit
1 kit
LC6070
SilverXpress Silver Staining Kit
1 kit
LC6100
SYPRO™ Ruby Protein Gel Stain
1L
S-12000
Large Gel Drying Kit
1 kit
NI2207
Quantity
Catalog No.
PageRuler™ Plus Prestained Protein Ladder
2 × 250 µL
26619
PageRuler Prestained Protein Ladder
2 × 250 µL
26616
NativeMark Unstained Protein Ladder
5 × 50 µL
LC0725
250 µL
LC5699
HiMark Unstained HMW Protein Standard
250 µL
LC5688
SeeBlue Plus2 Pre-Stained Standard
500 µL
LC5925
2 × 250 µL
10747-012
250 µL
LC5602
1 mL
LC5677
SimplyBlue SafeStain
™
Protein standards
™
™

HiMark Pre-Stained HMW Protein Standard

™
BenchMark™ Protein Ladder
MagicMark™ XP Western Protein Standard
Mark12 Unstained Standard
™
30
Novex™ Midi Gel System User Guide
Blotting Products
Quantity
Catalog no.
Nitrocellulose/Filter Paper Sandwich, 0.45 µm
16/pack
LC2006
Nitrocellulose/Filter Paper Sandwich, 0.2 µm
16/pack
LC2009
Invitrolon /Filter Paper Sandwich, 0.45 µm
16/pack
LC2007
Blotting Filter Paper (2.5 mm thick)
50/pack
LC2008
Blotting Roller
1 each
LC2100
Incubation Tray
8/pack
LC2102
WesternBreeze™ Chromogenic Kit, Anti-Mouse
1 kit
WB7103
WesternBreeze™ Chromogenic Kit Anti-Rabbit
1 kit
WB7105
WesternBreeze Chemiluminescent Kit, Anti-Mouse
1 kit
WB7104
WesternBreeze™ Chemiluminescent Kit, Anti-Rabbit
1 kit
WB7106
Quantity
Catalog no.
1 unit
PS0300
™
™
Power Supply
PowerEase 300W Power Supply
™
Novex™ Midi Gel System User Guide
31
Appendix E
Documentation and support
Obtaining support For the latest services and support information for all locations, go to thermofisher.com
At the website, you can:
32
•
Access worldwide telephone and fax numbers to contact Technical Support and Sales
facilities
•
Search through frequently asked questions (FAQs)
•
Submit a question directly to Technical Support ([email protected])
•
Search for user documents, SDSs, vector maps and sequences, application notes,
formulations, handbooks, certificates of analysis, citations, and other product support
documents
•
Obtain information about customer training
•
Download software updates and patches
Novex™ Midi Gel System User Guide
For support visit thermofisher.com/support or email [email protected]
thermofisher.com
11 January 2017
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