Bio-Rad Ready Gel Precast Gels for 2-D Electrophoresis User Manual
Application Guide
For Technical Service Call Your Local Bio-Rad Office
or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)
Catalog Number
161-0993
Ready Gel
®
Precast Gels
Table of Contents
Section 1 General Information.............................................................................................. 1
1.1 Introduction.......................................................................................................................................... 1
1.2 Ready Gel System Specifications......................................................................................................... 2
1.3 Ready Gel Comb Configurations.......................................................................................................... 2
Section 2 Setup and Basic Operation................................................................................... 3
2.1 Setting Up and Running Ready Gel Precast Gels................................................................................. 3
Section 3 SDS-PAGE.............................................................................................................7
3.1 Introduction.......................................................................................................................................... 7
3.2 Ready Gel Tris-HCl Gel Composition.................................................................................................... 8
3.3 Ready Gel Tris-HCl Gel Selection Guide............................................................................................... 8
3.4 SDS-PAGE Buffers............................................................................................................................... 9
3.5 Sample Preparation.............................................................................................................................. 9
3.6 Running Conditions.............................................................................................................................. 9
Section 4 Native PAGE..........................................................................................................10
4.1 Introduction..........................................................................................................................................10
4.2 Ready Gel Tris-HCl Gel Composition....................................................................................................10
4.3 Ready Gel Tris-HCl Gel Selection Guide ..............................................................................................11
4.4 Native PAGE Buffers.............................................................................................................................11
4.5 Sample Preparation.............................................................................................................................12
4.6 Running Conditions ............................................................................................................................ 12
Section 5 Peptide Analysis.................................................................................................. 11
5.1 Introduction......................................................................................................................................... 11
5.2 Ready Gel Tris-Tricine/Peptide Gel Composition..................................................................................11
5.3 Ready Gel Tris-Tricine/Peptide Gel Selection Guide............................................................................. 11
5.4 Tris-Tricine/Peptide Buffers.................................................................................................................. 12
5.5 Sample Preparation.............................................................................................................................12
5.6 Running Conditions............................................................................................................................ 12
Section 6 Isoelectric Focusing............................................................................................ 13
6.1 Introduction......................................................................................................................................... 13
6.2 Ready Gel IEF Gel Composition ......................................................................................................... 13
6.3 Ready Gel IEF Gel Selection Guide......................................................................................................13
6.4 IEF Buffers...........................................................................................................................................14
6.5 Sample Preparation.............................................................................................................................14
6.6 Running Conditions............................................................................................................................. 14
Section 7 Protease Analysis by Zymogram PAGE................................................................15
7.1 Introduction..........................................................................................................................................15
7.2 Ready Gel Zymogram Gel Composition...............................................................................................15
7.3 Ready Gel Zymogram Gel Selection Guide..........................................................................................15
7.4 Zymogram Gel Buffers.........................................................................................................................16
7.5 Sample Preparation.............................................................................................................................16
7.6 Running Conditions............................................................................................................................. 16
Section 8 Nondenaturing Nucleic Acid PAGE...................................................................... 17
8.1 Introduction......................................................................................................................................... 17
8.2 Ready Gel TBE Gel Composition.........................................................................................................17
8.3 Ready Gel TBE Gel Selection Guide....................................................................................................17
8.4 Nondenaturing Nucleic Acid PAGE Buffers.......................................................................................... 18
8.5 Sample Preparation.............................................................................................................................18
8.6 Running Conditions............................................................................................................................. 18
Section 9 Denaturing Nucleic Acid PAGE............................................................................19
9.1 Introduction......................................................................................................................................... 19
9.2 Ready Gel TBE-Urea Gel Composition................................................................................................ 19
9.3 Ready Gel TBE-Urea Gel Selection Guide........................................................................................... 19
9.4 TBE-Urea Buffers................................................................................................................................ 20
9.5 Sample Preparation............................................................................................................................ 20
9.6 Running Conditions............................................................................................................................ 20
Section 10 Detection.......................................................................................................... 21
10.1 SDS-PAGE and Native PAGE Detection...................................................................................... 21–22
10.2 Peptide Gel Staining......................................................................................................................... 23
10.3 IEF Gel Staining................................................................................................................................ 24
10.4 Zymogram Gel Staining.................................................................................................................... 25
10.5 TBE Gel Staining.............................................................................................................................. 26
10.6 TBE-Urea Gel Staining...................................................................................................................... 26
Section 11 Stock and Staining Solutions.......................................................................27–31
11.1 Stock Solutions................................................................................................................................. 27
11.2 Protein Staining Solutions............................................................................................................ 28–29
11.3 Peptide Staining Solutions................................................................................................................ 29
11.4 Zymogram Staining Solutions ...........................................................................................................30
11.5 Nucleic Acid Staining Solutions ........................................................................................................ 31
Section 12 Troubleshooting........................................................................................... 32–33
Section 13 Ordering Information......................................................................................... 34
13.1 Ready Gel Precast Gels ....................................................................................................................34
13.2 Ready Gel Accessories......................................................................................................................35
13.3 Buffers...............................................................................................................................................36
13.4 Detection Reagents........................................................................................................................... 37
13.5 Blotting Membranes.......................................................................................................................... 38
13.6 Protein and DNA Standards.............................................................................................................. 38
13.7 Equipment.........................................................................................................................................38
Section 1
General Information
1.1 Introduction
Ready Gel
®
precast gels greatly simplify polyacrylamide gel electrophoresis. They are specifically for use with
the Mini-PROTEAN Systems (Mini-PROTEAN Tetra, Mini-PROTEAN-III and Mini-PROTEAN Dodeca Cells).
Stringent production and quality control criteria, and the use of the highest quality reagents, ensure reproducible electrophoretic analysis with minimum effort. Every gel is checked during production for defects, and
each lot of gels is further tested by electrophoresis to verify quality.
Ready Gel precast gels come ready to use with preformed sample wells and a stacking gel when necessary.
Each Ready Gel Cassette is 8 x 10 cm (H x W) and 4.0 mm thick. Gel dimension is 6.8 x 8.6 cm (HxW) and
1.0 mm thick. Each gel is individually packaged in a leakproof pouch with an absorbent pad containing gel
buffer and 0.02% Sodium Azide.
Ready Gel precast gels are available for use in Tris-glycine (Tris-HCl and zymogram gels), Tris-Tricine, TBE,
TBE-urea, and IEF buffer systems. The Tris-HCl gels can be used for SDS-PAGE and non-SDS gel electrophoresis. The Tris-Tricine/peptide gels are optimized for peptide electrophoresis. The TBE gels are for use in
nucleic acid electrophoresis and can be used for native protein electrophoresis. TBE-urea gels provide
denaturing conditions for nucleic acids. Resolution of different size ranges of proteins or nucleic acids can
be obtained by choosing the correct gel.
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1.2 Mini-format gel System Specifications
Gel material Polyacrylamide
Gel dimensions 8.6 x 6.8 cm (W x L)
Gel thickness 1.0 mm
Resolving gel height 5.5 cm
Cassette dimensions 10 x 8.0 cm (W x L)
Cassette material Back (long): acrylic; front (short): glass
Comb material Polycarbonate
Total running buffer volume 700 ml for 2 gels, 1,000 ml for L. gels (Mini-PROTEAN Tetra Cell)
Storage conditions Store flat at 4ºC; DO NOT FREEZE
1.3 Ready Gel Comb Configurations
Comb Load Volume
9-well 30 µl
10-well 30 µl
10-well 50 µl
12-well 20 µl
15-well 15 µl
IPG 7 cm ReadyStrip
™
IPG strip
Prep 450 µl with one 15 µl reference well
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Section 2
Setup and Basic Operation Using Mini-PROTEAN Tetra Cell
2.1 Setting Up and Running Ready Gel Precast Gels
1. Each Ready Gel should be used immediately after it is removed from the storage pouch.
2. Remove the comb and gently rinse the wells with deionized water or running buffer.
3. Use the key knife or a razor blade to cut the tape at the bottom of the gel along the black “cut here” line.
It is helpful to cut all the way to the edge of the cassette where the pull tab begins.
4. Pull the tape tab along the cut line, up from the cassette and at an angle towards the comb end of the
gel.
Required materials:
• Clean and dry Mini-PROTEAN®Tetra cell tank
• Electrophoresis module (Electrode Assembly Module only for 1 or 2 gels; for 3 or 4 gels also use the
Companion Running Module)
• Running buffer (700 ml for 2 gels; 1000 ml for 4 gels)
• Ready Gel®precast gels or hand-cast gels
• PowerPac™Basic power supply
1. Assembly
3
Note: When running 2 gels only, use the Electrode Assembly (the one with the banana plugs), Not the
Companion Running Module (the one without the banana plugs). When running 4 gels, both the Electrode
Assembly and the Companion Running Module must be used, for a total of 4 gels (2 gels per assembly).
a. Set the clamping frame to the open position on a clean flat surface (see Figure 4a)
b. Place the first gel sandwich or gel cassette (with the short plate facing inward) onto the gel supports;
gel supports are molded into the bottom of the clamping frame assembly; there are two supports in
each side of the assembly. Note that the gel will now rest at a 30° angle, tilting away from the center
of the clamping frame. Please use caution when placing the first gel, making sure that
the clamping frame remains balanced and does not tip over. Now, place the second gel on
the other side of the clamping frame, again by resting the gel onto the supports. At this point there
will be two gels resting at an angle, one on either side of the clamping frame, tilting away from the
center of the frame (see Figure 4b).
Note: It is critical that gel cassettes are placed into the clamping frame with the short plate facing inward.
Also, the clamping frame requires 2 gels to create a functioning assembly, If an odd number of gels (1 or
3) is being run, you must use the buffer dam (see Figure 4b).
c. Using one hand, gently pull both gels towards each other, making sure that they rest firmly and
squarely against the green gaskets that are built into the clamping frame; make certain that the short
plates sit just below the notch at the top of the green gasket.
d. While gently squeezing the gel sandwiches or cassettes against the green gaskets with one hand
(keeping constant pressure and both gels firmly held in place), slide the green arms of the clamping
frame over the gels, locking them into place. Alternatively, you may choose to pick-up the entire
assembly with both hands, making sure that the gels do not shift, and simultaneously sliding both
arms of the clamping frame into place (see Figure 4c).
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The arms of the clamping frame push the short plates of each gel cassette up against the notch in
the green gasket, creating a leak-proof seal (check again to make certain that the short plates sit just
below the notch at the top of the green gasket). At this point, the sample wells can be washed-out
with running buffer, and sample can be loaded (Figure 4d).
Note: If running more than 2 gels, repeat steps 1a–d with the Companion Running Module.
Important Note: Do not attempt to lock the green arms of the clamping frame, without first ensuring
that the gel cassettes are perfectly aligned and stabilized against the notches on the green gaskets of the
module. To prevent the gels from shifting during the locking step, firmly and evenly grip them in place
against the core of the module with one hand.
CAUTION: When running 1 or 2 gels only, DO NOT
place the Companion Running Module
in the tank. Doing so will cause excessive heat generation and prevent
electrophoretic separation.
5
4a
4b
Fig. 4. Assembling the Mini-PROTEAN Tetra Cell Electrophoresis Module.
6
4c
4d
4e
Section 3
SDS-PAGE
3.1 Introduction
Ready Gel Tris-HCl gels provide a versatile system for the separation of proteins by molecular weight (SDS-PAGE
conditions) or charge to mass ratio (native conditions). (See section 4 for native PAGE applications and
protocols.) This is possible because Ready Gel Tris-HCl gels are made without SDS, allowing the sample
buffer and running buffer to determine the separation mechanism. Historically, SDS-PAGE systems contained
SDS in both the gel and the running buffer. Reproducible SDS-PAGE separations are performed in gels
lacking SDS provided the sample buffer and running buffers contain sufficient SDS to saturate the proteins
during electrophoresis. The recommended concentration of SDS is 2% in sample buffer and 0.1% in running
buffers.
SDS-PAGE uses discontinuous chloride and glycine ion fronts to form moving boundaries that stack and
then separate SDS-coated polypeptides by molecular weight. Protein samples are prepared in a reducing
denaturing sample buffer containing either 2-mercaptoethanol or dithiothreitol as the reducing reagent, and
heat and SDS are used to denature the proteins. 2-Mercaptoethanol and dithiothreitol eliminate protein
secondary structure by reducing disulfide bonds. SDS minimizes charge variability among proteins, giving
them the same charge to mass ratio and forcing them into rod-like shapes. This effectively eliminates the
effects of protein conformation and native charge density on the electrophoretic migration distance. Molecular
weight determinations are obtained by plotting the logarithm of protein molecular mass vs. the relative
mobility (Rf= distance migrated by protein/distance migrated by dye front).
7
3.2 Ready Gel Tris-HCl Gel Composition
Gel buffer 0.375 M Tris-HCl, pH 8.8
Cross-linker 2.6% C
Stacking gel 4% T, 2.6% C
Storage buffer 0.375 M Tris-HCl, pH 8.8
Shelf life 12 weeks
3.3 Ready Gel Tris-HCl Gel Selection Guide
Tris-HCl gels are available in a wide selection of single percentages and gradients for the separation of
proteins by SDS-PAGE.
Tris-HCl Gels Optimal Separation Tris-HCl Gradient Gels Optimal Separation
5% 100–250 kD 4–15% 20–250 kD
7.5% 40–200 kD 4–20% 10–200 kD
10% 30–150 kD 8–16% 20–120 kD
12% 20–120 kD 10–20% 10–100 kD
15% 10–100 kD
18% 10–50 kD
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3.4 SDS-PAGE Buffers
Running Buffer 1X Working Concentration 10x Stock
25 mM Tris Tris base 15.0 g
192 mM glycine Glycine 72.0 g
0.1% SDS SDS 5.0 g
to 500 ml with deionized water
Note: running buffer should be
~ pH 8.3. Do not adjust the pH.
Sample Buffer 2X Working Concentration
2X Stock
62.5 mM Tris-HCl, pH 6.8 0.5 M Tris-HCl, pH 6.8 1.0 ml
2% SDS 10% (w/v) SDS 1.6 ml
25% glycerol Glycerol 2.0 ml
0.01% Bromophenol Blue 1.0% Bromophenol Blue 0.08 ml
5% 2-mercaptoethanol 2-Mercaptoethanol 0.4 ml
or 350 mM DTT (added fresh) Deionized water 2.92
ml
8.0 ml
3.5 Sample Preparation
Determine the appropriate protein concentration of your sample based on the detection method and load
volume used. (See section 10.1 for approximate stain sensitivities.) Dilute 1 part sample with 1 part sample
buffer (see section 3.4) and heat at 95ºC for 5 min.
3.6 Running Conditions
Power conditions 200 V constant
Starting current: 50 mA/gel
Final current: 30 mA/gel
Run time 35 min
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