Ready Gel Precast Gels Application Guide ®

Ready Gel Precast Gels Application Guide ®
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®
Ready Gel
Precast Gels
Application Guide
Catalog Number
161-0993
For Technical Service Call Your Local Bio-Rad Office
or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)
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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.............................................................................................................
3.1 Introduction..........................................................................................................................................
3.2 Ready Gel Tris-HCl Gel Composition....................................................................................................
3.3 Ready Gel Tris-HCl Gel Selection Guide...............................................................................................
3.4 SDS-PAGE Buffers...............................................................................................................................
3.5 Sample Preparation..............................................................................................................................
3.6 Running Conditions..............................................................................................................................
7
7
8
8
9
9
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
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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
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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
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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
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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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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
10-well
10-well
12-well
15-well
IPG
Prep
30 µl
30 µl
50 µl
20 µl
15 µl
7 cm ReadyStrip™ IPG strip
450 µl with one 15 µl reference well
2
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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
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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).
4
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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.
4a
4b
5
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4d
4e
Fig. 4. Assembling the Mini-PROTEAN Tetra Cell Electrophoresis Module.
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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).
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Ready Gel Tris-HCl Gel Composition
Gel buffer
Cross-linker
Stacking gel
Storage buffer
Shelf life
3.3
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0.375 M Tris-HCl, pH 8.8
2.6% C
4% T, 2.6% C
0.375 M Tris-HCl, pH 8.8
12 weeks
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%
7.5%
10%
12%
15%
18%
100–250 kD
40–200 kD
30–150 kD
20–120 kD
10–100 kD
10–50 kD
4–15%
4–20%
8–16%
10–20%
20–250
10–200
20–120
10–100
8
kD
kD
kD
kD
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SDS-PAGE Buffers
Running Buffer
1X Working Concentration
25 mM Tris
192 mM glycine
0.1% SDS
10x Stock
Tris base
Glycine
SDS
to 500 ml with deionized water
15.0 g
72.0 g
5.0 g
Note: running buffer should be
~ pH 8.3. Do not adjust the pH.
Sample Buffer
3.5
2X Stock
0.5 M Tris-HCl, pH 6.8
10% (w/v) SDS
Glycerol
1.0% Bromophenol Blue
2-Mercaptoethanol
Deionized water
2X Working Concentration
62.5 mM Tris-HCl, pH 6.8
2% SDS
25% glycerol
0.01% Bromophenol Blue
5% 2-mercaptoethanol
or 350 mM DTT (added fresh)
1.0
1.6
2.0
0.08
0.4
2.92
8.0
ml
ml
ml
ml
ml
ml
ml
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
Run time
200 V constant
Starting current:
Final current:
35 min
50 mA/gel
30 mA/gel
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Section 4
Native PAGE
4.1
Introduction
Ready Gel Tris-HCl gels are made without SDS, allowing separation of protein in their native conformation.
The nonreducing and nondenaturing environment of native PAGE allows the detection of biological activity
and can improve antibody detection. Native PAGE can also be used to resolve multiple protein bands where
molecular mass separation by SDS-PAGE would reveal only one.
Native PAGE uses the same discontinuous chloride and glycine ion fronts as SDS-PAGE to form moving
boundaries that stack and then separate polypeptides by charge to mass ratio. Proteins are prepared in a
nonreducing nondenaturing sample buffer, which maintains the proteins’ secondary structure and native
charge density. Native PAGE is not suitable for accurate molecular weight determination due to the variability
of charge to mass ratio among different proteins.
4.2
Ready Gel Tris-HCl Gel Composition
Gel buffer
Cross-linker
Stacking gel
Storage buffer
Shelf life
0.375 M Tris-HCl, pH 8.8
2.6% C
4% T, 2.6% C
0.375 M Tris-HCl, pH 8.8, NaN3
12 weeks from the date of manufacture
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Ready Gel Tris-HCl Gel Selection
Native PAGE separates by charge to mass ratio, making individual protein migration protein dependent.
Optimal Tris-HCl gel percentages will have to be determined experimentally.
4.4
Native PAGE Buffers
Running Buffer
Working Concentration
25 mM Tris
192 mM glycine
10x Stock
Tris base
Glycine
to 500 ml with deionized water
15.0 g
72.0 g
87.0 g
Note: running buffer should be
~ pH 8.3. Do not adjust the pH.
Sample Buffer
2X Stock
0.5 M Tris-HCl, pH 6.8
Glycerol
1% Bromophenol Blue
Deionized water
2X Working Concentration
62.5 mM Tris-HCl, pH 6.8
25% glycerol
1% Bromophenol Blue
11
1.0 ml
2.0 ml
1.0 ml
4.92 ml
8.0 ml
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Sample Preparation
Determine the desired protein concentration and load volume of your sample based on the detection
method used. (See section 10.1 for approximate stain sensitivities). Sample preparation for native PAGE
applications requires special consideration. In the absence of SDS, the net charge of a polypeptide will be
determined by the pH of the sample buffer. Only polypeptides with a net negative charge will migrate into a
native PAGE Tris-HCl gel. Most polypeptides have an acidic or slightly basic pl (~3–8). These proteins can be
separated using a standard protocol by diluting 1 part sample with 1 part native sample buffer (see section
4.4; DO NOT HEAT SAMPLES).
Strongly basic peptides (pl >9) will have a net positive charge in a native PAGE Tris-HCl gel. In order for
polypeptides with a net positive charge to migrate into a native PAGE Tris-HCl gel, the polarity of the
electrodes must be changed by reversing the color-coded jacks when connecting to the power supply.
4.6
Running Conditions
Power conditions
Run time
200 V constant
Starting current:
Final current:
35 min
50 mA/gel
30 mA/gel
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Section 5
Peptide Analysis
5.1
Introduction
Ready Gel Tris-Tricine/peptide gels are optimized for separating peptides and proteins <10 kD. Superior
resolution of peptides is achieved by moving the peptide-SDS complexes more slowly through the gel. This
allows the faster moving SDS micelles, which normally interfere with peptide separations, to completely
separate from the peptides, allowing distinct peptide bands to resolve.
5.2
Ready Gel Tris-Tricine/Peptide Gel Composition
Gel buffer
Cross-linker
Stacking gel
Storage buffer
Shelf life
5.3
1.0 M Tris-HCl, pH 8.45
2.6% C
4% T, 2.6% C
1.0 M Tris-HCl, pH 8.45, NaN3
12 weeks from the date of manufacture
Ready Gel Tris-Tricine/Peptide Gel Selection Guide
Tris-Tricine/peptide gels are available in either a single percentage gel or a linear gradient gel.
Peptide Gel
16.5%
10–20%
Optimal Separation
15–30 kD
1–40 kD
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Tris-Tricine/Peptide Buffers
Running buffer
Working Concentration
100 mM Tris
100 mM Tricine
0.1% SDS
10x Stock
Tris base
Tricine
SDS
to 500 ml with deionized water
60.55 g
89.60 g
5.0 g
Note: Tricine running buffer should be
~ pH 8.25. Do not adjust the pH.
Sample Buffer
5.5
Working Concentration
200 mM Tris-HCl, pH 6.8
2% SDS
40% glycerol
0.04% Coomassie Blue G-250
2% 2-mercaptoethanol
or 350 mM DTT (Added fresh)
2X Stock
1.0 M Tris-HCl, pH 6.8
10% SDS
Glycerol
0.5% Coomassie Blue G-250
2-Mercaptoethanol
Deionized water
2.0
2.0
4.0
0.8
0.2
1.0
10.0
ml
ml
ml
ml
ml
ml
ml
Sample Preparation
Determine the appropriate protein concentration of your sample based on the detection method and load
volume used. (See section 10.2 for approximate stain sensitivities.) Dilute 1 part sample with 1 part sample
buffer and heat at 95ºC for 5 min.
5.6
Running Conditions
Power Conditions
Run Time
100 V constant
Starting current:
Final current:
30–35 mA/gel
15–20 mA/gel
100 min
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Section 6
Isoelectric Focusing
6.1
Introduction
Ready Gel IEF gels are cast with Bio-Rad’s Bio-Lyte® ampholytes, amphoteric molecules that set up a pH
gradient across the gels. Proteins migrate in IEF gels to their neutral isoelectric point (pI), where the protein
has zero net charge. Ready Gel IEF gels contain no denaturing agents, so all focusing is performed under
native conditions.
6.2
Ready Gel IEF Gel Composition
Gel buffer
Cross-linker
Stacking gel
Storage buffer
2% ampholyte, pH 3–10, 5–8
3.0% C
None
Deionized water, NaN3
Shelf life
26 weeks from the date of manufacture
6.3
Ready Gel IEF Gel Selection Guide
Ready Gel IEF gels are available in narrow and broad pH ranges.
IEF gel
pH Range
5–8
3–10
5–8
4–8.5
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IEF Buffers
Running buffer
Sample Buffer
6.5
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1x Cathode Buffer
20 mM lysine (free base)
20 mM arginine (free base)
5x Cathode Buffer
Lysine (free base)
Arginine (free base)
to 1 L with deionized water
14.50 g
17.42 g
1x Anode Buffer
7mM% phosphoric acid
10x Anode Buffer
Phosphoric acid
to 1 L with deionized water
4.2 ml
50% glycerol
Sample Preparation
Determine the appropriate protein concentration of your sample based on the detection method and load
volume used. (See section 10.3 for approximate stain sensitivities.) Dilute 1 part sample with 1 part sample
buffer.
6.6
Running Conditions
Power conditions
Run time
Stepwise
100 V constant
250 V constant
500 V constant
Starting current:
Final current:
60 min
60 min
30 min
5–15 mA/gel
5–15 mA/gel
150 min
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Section 7
Protease Analysis by Zymogram PAGE
7.1
Introduction
Ready Gel zymogram gels are used to test for proteolytic activity when performing protein characterizations.
Gels are cast with gelatin or casein, which act as substrates for proteases that are separated on the gel.
Proteases are detected by renaturing the enzyme followed by a development period in which the protease
breaks down the substrate. Zymogram gels are stained with Coomassie Blue R-250, which stains the
substrate while leaving clear areas around active proteases.
7.2
Ready Gel Zymogram Gel Composition
Gel buffer
Cross-linker
Stacking gel
Storage buffer
Shelf life
7.3
0.375 M Tris-HCl, pH 8.6
2.6% C
4% T, 2.6% C
0.375 M Tris-HCl, pH 8.6, 0.2% NaN3
12 weeks from the date of manufacture
Ready Gel Zymogram Gel Selection Guide
Ready Gel zymogram gels are available with either gelatin or casein as substrate and should be selected
based on their substrate and separation range.
Zymogram Gel
Optimal Separation
10% zymogram gel with gelatin
12% zymogram gel with casein
30–150 kD
20–120 kD
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Zymogram Buffers
Running buffer
Working Concentration
25 mM Tris
192 mM glycine
0.1% SDS
10x Stock
Tris base
Glycine
SDS
to 500 ml with deionized water
15.0 g
72.0 g
5.0 g
Note: running buffer should be
~pH 8.3. Do not adjust the pH.
Sample Buffer
7.5
Working Concentration
62.5 mM Tris-HCl, pH 6.8
4% SDS
25% glycerol
0.01% Bromophenol Blue
2X Stock
0.5 M Tris-HCl, pH 6.8
10% SDS
Glycerol
1% Bromophenol Blue
Deionized water
1.25
4.0
2.5
0.1
2.15
10
ml
ml
ml
ml
ml
ml
Sample Preparation
Determine the appropriate protein concentration of your sample based on the detection method and load
volume used. (See section 10.4 for approximate stain sensitivities.) Dilute 1 part sample buffer with 1 part
Zymogram sample buffer. Dry samples can be dissolved directly in sample buffer. Do not heat.
7.6
Running Conditions
Power conditions
Run time
100 V constant
Starting current:
Final current:
10–15 mA/gel
6 mA/gel
90 min
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Section 8
Nondenaturing Nucleic Acid PAGE
8.1
Introduction
Ready Gel TBE gels are ideal for separating small dsDNA fragments, especially PCR products. The uniform
nature of DNA molecules provides samples with near-uniform charge to mass ratio, allowing nondenaturing
nucleic acid PAGE to separate dsDNA by mass using a continuous TBE buffer system.
8.2
Ready Gel TBE Gel Composition
Gel buffer
Cross-linker
Stacking gel
Storage buffer
Shelf life
8.3
89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3
3.3% C
4% T, 3.3% C
89 mM Tris, 89 mM boric acid, 2 mM EDTA, NaN3
12 weeks from the date of manufacture
Ready Gel TBE Gel Selection Guide
Ready Gel TBE gels are available in a selection of single percentages and gradients for the separation of
dsDNA.
TBE Gels
Optimal Separation
TBE Gradient Gels
Optimal Separation
5%
10%
15%
200–2,000 bp
50–1,500 bp
20–1,000 bp
4–20%
10–2,000 bp
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Nondenaturing Nucleic Acid PAGE Buffers
Running Buffer
Working Concentration
50 mM Tris
89 mM boric acid
5 mM EDTA
10x Stock
Tris base
Boric acid
0.5 M EDTA (pH 8.0)
to 500 ml with deionized water
0.06 g
27.5 g
0.1 ml
Note: TBE running buffer should be
~ pH 8.3. Do not adjust the pH.
Sample Buffer
8.5
2X Working Concentration
50 nM EDTA
25% glycerol
0.2% Bromophenol Blue
0.2% Xylene Cyanole FF
Tris Base
0.06 g
0.5 M EDTA
0.1 ml
Glycerol
2.5 ml
1% Bromophenol Blue
2.0 ml
1% Xylene Cyanole FF
2.0 ml
Make up to 10 ml with deionized water
Sample Preparation
Determine the desired DNA concentration of your sample based on the detection method used. (See
section 10.5 for approximate stain sensitivities.) Dilute 1 part sample with 4 parts sample buffer (see section
8.4).
8.6
Running Conditions
Power conditions
Run time
100 V constant
Starting current:
Final current:
13 mA/gel
11 mA/gel
45–105 min
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Section 9
Denaturing Nucleic Acid PAGE
9.1
Introduction
Ready Gel TBE-urea gels are ideal for separating small ssDNA and RNA fragments. Applications include
oligonucleotide analysis, RNase protection assays, and northern blotting.
9.2
Ready Gel TBE-Urea Gel Composition
Gel buffer
Cross-linker
Stacking gel
Storage buffer
Shelf life
9.3
89 mM Tris, 89 mM boric acid, 2 mM EDTA, 7 M urea, pH 8.3
3.3% C
4% T, 3.3% C
89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3, NaN3
8 weeks from the date of manufacture
Ready Gel TBE-Urea Gel Selection Guide
Ready Gel TBE-urea gels are available in a range of single percentage gels.
TBE-Urea
5%
10%
15%
Optimal Separation
50–1,000 bases
25–300 bases
10–50 bases
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TBE-Urea Buffers
Running Buffer
Sample Buffer
9.5
9:13 AM
Working Concentration
89 mM Tris
89 mM boric acid
2 mM EDTA
10x Stock
Tris base
Boric acid
0.5 M EDTA (pH 8.0)
to 500 ml with deionized water
Working Concentration
89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0
12% Ficoll
0.01% Bromophenol Blue
0.02% Xylene Cyanole FF
7 M urea
54.0 g
27.5
20.0 ml
10x TBE
1.0 ml
Ficoll
1.2 g
Urea
4.2 g
1% Bromophenol blue
0.1 ml
1% Xylene Cyanole FF
0.2 ml
0.5 M EDTA
0.02 ml
Make up to 10 ml with deionized water
Sample Preparation
Determine the desired ssDNA or RNA concentration for your sample based on the detection method used.
(See section 10.6 for appropriate stain sensitivities.) Dilute 1 part sample with 1 part TBE-urea sample buffer.
Dry samples can be dissolved directly in sample buffer. Heat to 70–90°C 4 min before loading.
9.6
Running Conditions
Power conditions
Run time
200 V constant
Starting current:
Final current:
15 mA/gel
10 mA/gel
40–70 min
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Section 10
Detection
10.1
SDS-PAGE and Native PAGE Detection
Total Protein Gel Stains
Method
Sensitivity
Optimal Protein Load
Advantages
Disadvantages
Coomassie Blue R-250
36–47 ng
Bio-Safe™ Coomassie stain 8–28 ng
~0.5 µg/band
Laboratory standard
Requires MeOH
~0.5 µg/band
Nonhazardous, uses
no MeOH
More steps than
Coomassie Blue R-250
Copper stain
6–12 ng
~0.2 µg/band
Fast, reversible stain
be photographed;
SDS-PAGE only
Negative stain, must
Zinc stain
6–12 ng
~0.2 µg/band
High-contrast, fast,
reversible stain
SDS-PAGE only
Negative stain, must
be photographed;
Silver Stain™ Plus kit
0.6–1.2 ng
~0.01 µg/band
Simple, robust, mass
spectrometry compatible
Will not stain
glycoproteins
Silver stain
0.6–1.2 ng
~0.01 µg/band
Stains complex
proteins, i.e., glycoproteins
and lipoproteins
Not mass spectrometry
compatible
SYPRO Orange protein
stain
4–8 ng
~0.2 µg/band
Will not stain nucleic
acids; mass spectrometry
compatible
Optimization required
for maximum
sensitivity
SYPRO Ruby protein gel
stain
1–10 ng
~0.2 µg/band
Broad dynamic range,
simple robust protocol
maximum sensitivity
Requires imaging
instrument for
Flamingo Fluorescent
gel stain
0.25–0.5 ng
~0,2 ng/band
Broad dynamic range
mass spec compatible
Requires imaging instrument
for maximum sensitivity
23
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Total Protein Blot Stains
Method
Sensitivity
Optimal Protein Load
Advantages
Disadvantages
SYPRO Ruby protein blot
stain
2–8 ng
~0.2 µg/band
Multiple step protocol; Requires
imaging instrument for
immunological procedures
Compatible with mass
spectrometry, Edman-based
sequencing, and standard
maximum sensitivity
Colloidal gold stain
1 ng
~0.1 µg/band
Sensitive, one step
Not compatible with nylon
membranes
Enhanced colloidal
gold detection kit
10–100 pg
~0.1 µg/band
Increases sensitivity of
colloidal gold stain
Multiple steps
AmidoBlack 10B
100–1,000 ng
~5 µg/band
Standard membrane
stain, economical
Low sensitivity
Method
Sensitivity
Optimal Protein Load
Advantages
Disadvantages
4CN colorimetric (HRP)
500 pg
~0.25 µg/band
Fast detection
Results may fade
DAB colorimetric (HRP)
Immunoblot Detection
500 pg
~0.25 µg/band
Fast detection
Contains toxic chemicals
Opti-4CN colorimetric (HRP) 100 pg
~0.05 µg/band
Color does not fade
More expensive than 4CN
Amplified Opti-4CN
colorimetric (HRP)
10 pg
~0.005 µg/band
High sensitivity, low
background
Amplification requires
additional steps
BCIP/NBT colorimetric
(AP)
100 pg
~0.05 µg/band
Sensitive, multiple antigen
May detect endogenous
enzyme activity
Amplified alkaline
phosphatase
Immun-Star™
10 pg
~0.005 µg/band
High sensitivity
Amplification requires
additional steps
10 pg
~0.005 µg/band
Long-lasting signal,
short and multiple
exposures possible
Requires visualization
on film or instrumentation
1–3 pg
~0.005 µgband
Intensifies signal
output, very sensitive
Requires visualization on
film or instrumentation
~0.005 µgband
long-lasting signal
short and multiple
exposures possible
Requires visualzation on
on film or instrumentation
chemiluminescent (AP)
Immun-Star™
chemiluminescent (hrp)
Immun-Star WesternC (HRP) 10 fg
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Peptide Gel Staining
Peptides and small proteins are prone to diffusion and loss during staining. The following protocol uses a
fixation step to prevent sample loss and is suitable for detection of bands as low as 10–20 ng.
Fixative Solution
40% methanol
10% acetic acid
Coomassie Brilliant Blue G-250 Stain Solution
0.025% Coomassie Blue G-250
10% acetic acid
Destain Solution
10% acetic acid
Place gels in fixative solution and equilibrate for 30 min. Stain gels with Coomassie Brilliant Blue G-250 stain
solution for 1 hr. Stain should only be used once. Reuse of stain could result in loss of sensitivity. Destain
gels 3 times for 15 min or until the desired background is achieved. Some peptides may not be completely
fixed and may diffuse out of the gels if fixing and staining times are greatly exceeded.
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10.3 IEF Gel Staining
Samples on IEF gels can be detected using multiple methods. Use the following table as a guide to select
an appropriate staining method.
Method
Sensitivity
Optimal Protein Load
Advantages
IEF stain
40–50 ng
~0.5 µg/band
Simple, no fixation required
Requires MeOH
Silver Stain Plus kit
0.6–1.2 ng
~0.01 µg/band
Simple, robust, mass
spectrometry compatible
Requires TCA
fixation
Silver stain
0.6–1.2 ng
~0.01 µg/band
Stains complex
proteins, i.e. glycoproteins
and lipoproteins
Requires TCA
fixation
26
Disadvantages
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10.4 Zymogram Gel Staining
Prior to staining zymogram gels, sample proteases must be first renatured and allowed to break down the
substrate contained in the gel. The following protocol provides basic guidelines for detection. Optimal results
should be determined empirically.
Renaturing Solution
2.5% Triton X-100
Development Solution
50 mM Tris
200 mM NaCl
5 mM CaCl2 (anhydrous)
0.02% Brij-35
Adjust to pH 7.5
Staining Solution
40% methanol
10% acetic acid
0.5% Coomassie Blue R-250
Destaining Solution
40% methanol
10% acetic acid
Proteins must be renatured first by placing the gels in renaturing solution for 30 min at room temperature.
Incubate gels in development solution at 37ºC for a minimum of 4 hr. Highest sensitivity is typically achieved
with overnight incubation. Optimal results should be determined empirically. Stain gels with Coomassie
Brilliant Blue R-250 staining solution for at least 1 hr at room temperature. Destain until clear bands appear
against the blue background, approximately
27
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30–60 min.
10.5 TBE Gel Staining
Use the following table as a guide to select an appropriate staining method.
Method
Sensitivity
Optimal Protein Load
Advantages
Disadvantages
Ethidium bromide
50 ng
~0.10 µg/band
Classic fluorescent DNA stain
Carcinogenic
Silver stain
1.0–2.0 ng
~0.5 µg/band
More sensitive than
ethidium bromide
Multiple steps
10.6
TBE-Urea Gel Staining
Samples on denaturing nucleic acid gels can be detected using multiple methods. Use the following table as
a guide to select an appropriate staining method.
Method
Ethidium bromide
Sensitivity
Optimal Protein Load
Advantages
Disadvantages
50 ng
~0.10 µg/band
Classic fluorescent DNA stain
Carcinogenic
Radiant Red
10 ng
~0.10 µg/band
Fast single-step protocol
RNA and ssDNA
only
Silver stain
1.0–2.0 ng
~0.5 µg/band
More sensitive than
ethidium bromide
Multiple steps
®
28
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Section 11
Stock and Staining Solutions
11.1
Stock Solutions
0.5 M Tris-HCl, pH 6.8
6.06 g Tris base
~60 ml deionized water
Adjust to pH 6.8 with HCl. Make to 100 ml with deionized water and store at 4°C.
10% SDS
Dissolve 1 g SDS in water with gentle stirring and bring to 10 ml with deionized water.
1% Bromophenol Blue
Dissolve 0.1 g of Bromophenol Blue in 10 ml deionized water with gentle stirring.
1% Xylene Cyanole FF
Dissolve 0.1 g of Xylene Cyanole FF in 10 ml deionized water with gentle stirring.
0.5 M EDTA
18.6 g of EDTA
~ 50 ml of deionized water
Adjust to pH 8.0 with 1 N NaOH. Make to 100 ml with deionized water and store at 4°C.
29
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Protein Staining Solutions
Coomassie Blue R-250 Staining Solution (0.1%)
Final Concentration
Methanol
400 ml
40%
Acetic acid
100 ml
10%
Coomassie Blue R-250
1.0 g
0.1%
Deionized water
500 ml
Dissolve Coomassie R-250 in methanol/acetic acid. Add deionized water to a final volume of 500 ml.
Coomassie Blue R-250 Destaining Solution
Final Concentration
Methanol
400 ml
40%
Acetic acid
100 ml
10%
Deionized water
500 ml
Silver Staining
See Bio-Rad’s silver stain (catalog #161-0443) or Silver Stain Plus kit (catalog #161-0449) instructions.
IEF Staining Solution
Final Concentration
Isopropyl alcohol
Acetic acid
Coomassie Blue R-250
Crocein Scarlet
Deionized water
270 ml
100 ml
0.4 g
0.5 g
630 ml
27%
10%
0.04%
0.05%
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IEF Destaining Solution
Final Concentration
Methanol
400 ml
40%
Acetic acid
100 ml
10%
Deionized water
500 ml
11.3
Peptide Staining Solutions
Fixative Solution
Final Concentration
Methanol
400 ml
40%
Acetic acid
100 ml
10%
Deionized water
500 ml
Coomassie Blue G-250 Staining Solution (0.025%)
Final Concentration
Acetic acid
100 ml
10%
Coomassie Blue G-250
0.25 g
0.025%
Deionized water
900 ml
Coomassie Blue G-250 Destaining Solution
Final Concentration
Acetic acid
100 ml
Deionized water
900 ml
10%
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Zymogram Staining Solutions
Renaturation Buffer
Final Concentration
Triton X-100
Deionized water
to 1 L with deionized water
25 g
900 ml
2.5 %
Development Solution
Final Concentration
Tris base
NaCl
CaCl2 (anhydrous)
30% Brij-35
6.06
11.7
0.56
0.67
g
g
g
ml
50 mM
200 mM
5 mM
0.02%
Dissolve in 900 ml deionized water, adjust to 7.5 with 6 N HCl, make to 1 L with deionized water.
Staining Solution
Final Concentration
Methanol
400 ml
Acetic acid
100 ml
Coomassie Blue R-250
5g
Deionized water
500 ml
Dissolve Commassie R-250 in Methnaol/acetic
40%
10%
0.5%
acid. Add deionized water to final volume of 500 ml.
32
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Nucleic Acid Staining Solutions
Ethidium Bromide Staining
Use Bio-Rad’s ethidium bromide tablets or ethidium bromide solutions (catalog #161-0430 or 161-0443) for nucleic acid staining
solutions.
Silver Staining
See instructions for Bio-Rad’s silver stain, catalog #161-0443, or Silver Stain Plus kit, catalog #161-0449.
Radiant® Red Staining
Use Radiant Red stain, catalog #170-3122, for RNA staining.
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Section 12
Troubleshooting
Improper storage of Ready Gel precast gels can produce numerous artifacts. Gels should be stored flat at
4ºC. Avoid freezing or prolonged storage above 4ºC. If you suspect your gels have been stored improperly,
DO NOT USE THEM.
Problem
Possible Cause
Solution
Samples do not migrate into gel
Tape at the bottom of the cassette
not removed
Remove tape
Insufficient buffer in integral buffer chamber
Fill buffer chamber with 125 ml
running buffer
Insufficient lower electrode buffer
Fill both halves of the lower buffer tank
with sufficient running buffer
Electrical disconnection
Check electrodes and connections
Bands “smile” across gel, band pattern
curves upward at both sides of the gel
Excess heating of gel
Check buffer composition
Completely fill both halves of the lower
buffer tank with sufficient running buffer
Do not exceed recommended running
conditions
Skewed or distorted bands, lateral
band spreading
Excess salt in samples
Remove salts from sample by dialysis or
desalting column prior to sample
preparation
Insufficient sample buffer or wrong formulation
Check buffer composition and dilution
instructions
Problem
Possible Cause
Solution
Vertical streaking
Overloaded samples
Dilute sample
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Selectively remove predominant protein in
the sample
Sample precipitation
Centrifuge samples to remove particulates
prior to sample loading
Gels run too fast, provide poor resolution,
and gel temperature is too high
Running buffer is too concentrated
Check buffer composition
Artifact bands at ~60–70 kD
Possible skin keratin contamination
Clean all dishware and wear gloves
while handling and loading gel
Filter all solutions through nitrocellulose
Use 10% iodoacetamide to eliminate
keratin bands
35
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Section 13
Ordering Information
13.1
Ready Gel Precast Gels
Ready Gel Tris-HCl
Gels
10-Well
30 µl
15-Well
15 µl
5% Tris-HCl
7.5% Tris-HCl
10% Tris-HCl
12% Tris-HCl
15% Tris-HCl
18% Tris-HCl
4–15% Tris-HCl
4–20% Tris-HCl
8–16% Tris-HCl
10–20% Tris-HCl
161-1210
161-1100
161-1101
161-1102
161-1103
161-1216
161-1104
161-1105
161-1222
161-1106
161-1211
161-1118
161-1119
161-1120
161-1121
161-1217
161-1122
161-1123
161-1223
161-1124
Prep Well
450 µl
161-1136
161-1137
161-1138
161-1139
161-1140
161-1141
161-1142
10-Well
50 µl
12-Well
20 µl
161-1213
161-1154
161-1155
161-1156
161-1157
161-1219
161-1158
161-1159
161-1225
161-1160
161-1214
161-1172
161-1173
161-1174
161-1175
161-1220
161-1176
161-1177
161-1226
161-1178
161-1163
161-1164
161-1181
161-1182
161-1232
9-Well
30 µl
IPG Comb
7 cm IPG Strip
161-1191
161-1390
161-1391
161-1194
161-1392
161-1393
161-1394
161-1395
Ready Gel TBE Precast Gels
5%, TBE
10%, TBE
15%,TBE
4–20%, TBE
161-1109
161-1110
161-1228
161-1234
161-1127
161-1128
161-1229
161-1235
—
—
—
—
161-1237
Ready Gel Tris-Tricine/Peptide Precast Gels
16.5% Tris-Tricine/Peptide
161-1107
10–20% Tris-Tricine/Peptide 161-1108
161-1125
161-1126
161-1143
161-1144
36
161-1161
161-1162
161-1179
161-1180
161-1197
161-1198
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Ready Gel IEF Precast Gels
IEF pH 3–10
IEF pH 5–8
161-1111
161-1112
161-1129
161-1165
Ready Gel Zymogram Precast Gels
10% Zymogram, gelatin
161-1113
12.5% Zymogram, casein 161-1114
161-1131
—
—
161-1167
161-1168
161-1185
Ready Gel TBE-Urea Precast Gels
5% TBE-Urea
10% TBE-Urea
15% TBE-Urea
161-1115
161-1116
161-1117
161-1133
161-1134
161-1135
—
161-1189
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13.2 Buffers
Premixed Running Buffers
Premixed Sample Buffers
161-0732
161-0772
161-0734
161-0771
161-0744
161-0761
161-0762
161-0733
161-0770
161-0765
161-0766
161-0737
161-0738
161-0739
161-0763
161-0764
161-0767
161-0768
10x Tris/Glycine/SDS, 1 L
10x Tris/Glycine/SDS, 5 L
10x Tris/Glycine, 1 L
10x Tris/Glycine, 5 L
10x Tris/Tricine/SDS, 1 L
10x IEF Anode Buffer, 250 ml
10x IEF Cathode Buffer, 250 ml
10x Tris/Boric Acid/EDTA, 1 L
10x Tris/Boric Acid/EDTA, 5 L
Zymogram Renaturation Buffer, 125 ml
Zymogram Development Buffer, 125 ml
* Requires addition of 2-mercaptoethanol or DTT
Individual Reagents
161-0719
161-0716
161-0717
161-0718
161-0724
161-0301
161-0710
161-0610
161-0611
161-0404
Laemmli Sample Buffer, 30 ml*
Native Sample Buffer, 30 ml
Tricine Sample Buffer, 30 ml
IEF Sample Buffer, 30 ml
Zymogram Sample Buffer, 30 ml
Nucleic Acid Sample Buffer, 5x, 10 ml
TBE-Urea Sample Buffer, 30 ml
Tris, 1 kg
Tris, 500 g
Glycine, 250 g
Glycine, 1 kg
Glycine, 2 kg
SDS, 100 g
2-Mercaptoethanol, 25 ml
Dithiothreitol, 1 g
Dithiothreitol, 5 g
Bromophenol Blue, 10 g
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13.3 Detection Reagents
Total Protein Gel Stains
Total Protein Blot Stains
161-0436
161-0438
161-0400
161-0786
Coomassie Blue R-250 Stain Solution, 1 L
Coomassie Blue R-250 Destain Solution, 1 L
Coomassie Brilliant Blue R-250, 10 g
Bio-Safe Coomassie Stain, 1 L
170-3127
170-6527
170-6517
161-0402
161-0470
161-0440
Copper Stain and Destain Kit
Zinc Stain and Destain Kit
161-0449
161-0443
Silver Stain Plus Kit
Bio-Rad Silver Stain Kit
170-3120
170-3125
161-0490
161-0491
161-0492
SYPRO Orange Protein Stain, 500 µl
SYPRO Ruby Protein Gel Stain, 1 L
Flamingo Fluorescent Gel Stain (10x), 20 ml
Flamingo Fluorescent Gel Stain (10x), 100 ml
Flamingo Fluorescent Gel Stain (10x), 500 ml
Immunoblot Detection
170-6431
170-6535
170-8238
170-8235
170-6432
170-6412
170-5012
HRP Conjugate Substrate Kit, 4CN
HRP Color Development Reagent, DAB
Amplified Opti-4CN Kit
Opti-4CN Substrate Kit
BCIP/NBT AP Conjugate Substrate Kit
Amplified Alkaline Phosphatase Kit
Immun-Star™ Substrate Pack
39
SYPRO Ruby Protein Blot Stain, 200 ml
Colloidal Gold Total Protein Stain, 500 ml
Enhanced Colloidal Gold Detection Kit
Amido Black 10B, 25 g
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13.4
Blotting Membranes
162-0232
162-0233
162-0234
162-0235
162-0236
162-0237
0.2 µm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack
0.2 µm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 50 pack
0.45 µm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack
0.45 µm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 50 pack
Sequi-Blot PVDF/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack
Sequi-Blot PVDF/Filter Paper Sandwich, 8.5 x 13.5 cm, 50 pack
13.5
Protein and DNA Standards
161-0363
161-0373
161-0324
161-0326
161-0310
161-0375
161-0370
Precision Plus Protein™ Unstained Standards (10–250 kD), 1,500 µl, 150 applications
Precision All Blue Prestained Standards (10–250 kD), 500 µl, 50 applications
Kaleidoscope™ Prestained Standards, 500 µl, 50 applications
Polypeptide SDS-PAGE Standards (1.4–26.6 kD), 200 µl, 400 applications
IEF Standards, pI range 4.45–9.6, 250 µl, 500 applications
Precision Plus Protein™ Kalaidoscope Standards 500 µl, 50 applications
Precision Plus Protein™ WesternC Standards, 250 µl, 50 applications
170-8351
170-8352
170-8353
170-8200
165-8004
20 bp EZ Load™ Molecular Ruler (20–1,000 bp), 50 µg, 100 applications
100 bp EZ Load Molecular Ruler (100–1,000 bp), 25 µg, 100 applications
100 bp PCR EZ Load Molecular Ruler (100–3,000 bp), 40 µg, 100 applications
AmpliSize® Molecular Ruler (50–2,000 bp), 25 µg, 50 applications
Mini-PROTEAN Tetra Cell for Ready Gel Precast gels
13.6
Equipment
165-3302
170-3930
Mini-PROTEAN 3 Electrophoresis Module
Mini Trans-Blot® Electrophoresis Transfer Cell
Brij is a trademark of ICI Americas, Inc. Coomassie is a trademark of Imperial Chemical Industries PLC. Ficoll is a trademark of Amersham
Pharmacia Biotech. SYPRO is a trademark of Molecular Probes, Inc. Bio-Rad is licensed to sell SYPRO products for research use only,
under US Patent 5,616,502. Triton is a trademark of Union Carbide.
40
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Page A
Catalog Number
161-0993
Bio-Rad
Laboratories, Inc.
Life Science
Group
Bulletin 0000
US/EG
Web site www.bio-rad.com USA (800) 4BIORAD Australia 02 9914 2800
Austria (01) 877 89 01 Belgium 09-385 55 11 Brazil 55 21 507 6191
Canada (905) 712-2771 China (86-21) 63052255
Czech Republic (420) 2-4141 0532 Denmark 45 44 52-1000
Finland 358 (0)9 804 2200 France 01 47 95 69 65 Germany 089 318 84-177
Hong Kong 852-2789-3300 India (91-124) 6398112/113/114, 6450092/93
Israel 03 951 4127 Italy 39 02 216091 Japan 03-5811-6270
Korea 82-2-3473-4460 Latin America 305-894-5950
Mexico 52 5 534 2552 to 54 The Netherlands 0318-540666
New Zealand 64-9-4152280 Norway 47-23-38-41-30 Poland (48) 22-8126 672
Portugal 351-21-472-7700 Russia 7 095 721 1404 Singapore 65-2729877
South Africa 00 27 11 4428508 Spain 34 91 590 5200
Sweden 46 (0)8-55 51 27 00 Switzerland 061 717-9555 United Kingdom 0800-181134
Rev A
00-000
0000
Sig 1001
LIT188 Rev H
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