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.

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

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

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).

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.

Fig. 4. Assembling the Mini-PROTEAN Tetra Cell Electrophoresis Module.

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).

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

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

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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