| Contributor: |
The Laboratory of Jasper Rine at the University of California, Berkeley
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| This protocol is a general method for the preparation of gradient polyarcylamide gels. The procedure requires access to and a general knowledge of a gradient maker. |
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A. Assembling the Gel Molds and Preparing the SDS-Polyacrylamide Solutions.
1. Use a clean mold and clean backing. Remove the clamps and the cover from a gel casting apparatus. Insert the two blocks that represent the width of the gel (usually 0.75 mm).
2. Mount the assemble gel casting unit upright to facilitate pouring the gel.
3. Prepare the appropriate percentage Polyacrylamide solution for the resolving gel (see Hints #1, #2 and #3):
To prepare Low Percentage (7.5%) SDS-Polyacrylamide Gel Solution add
25 ml of 30%:0.8% Acrylamide:Bis-Acrylamide (CAUTION! see Hint #4)
25 ml of 4X Lower Tris Buffer (or buffer of your choice)
50 ml of ddH2O
Add 200 μl of 10% APS (do not add the TEMED just yet)
To prepare High Percentage (15%) SDS-Polyacrylamide Gel Solution add
50 ml of 30%:0.8% Acrylamide:Bis-Acrylamide (CAUTION! see Hint #4)
25 ml of 4X Lower Tris Buffer (or buffer of your choice)
25 ml of ddH2O
Add 200 μl of 10% APS (do not add the TEMED just yet)
B. Preparing the Gradient Maker and Pouring the Resolving Gel.
1. Attach tubing to a low pressure pump and align the pump such that fluid will flow from a 1X Lower Tris Buffer tank through the pump and then into the gradient maker's inlet port (see Hint #5).
2. Attach tubing to run from the gradient maker outlet port to the top of the gel. Tape the tubing securely at the top of the glass molding of the gel directly in the center such that the fluid will flow from the tubing down the inside of the glass.
3. Work quickly through the next steps.
4. Add 50 μl of TEMED to each of the Polyacrylamide Solutions (see Hint #6).
5. Load the Low Percentage (7.5%) Polyacrylamide Solution into the first chamber of the gradient maker (see Hint #5)
6. Load the High Percentage (15%) Polyacrylamide Solution into the second chamber of the gradient maker.
7. Initiate flow on the pump (see Hint #7). Stop the pump when the solution is approximately 3 to 4 cm from the top of the glass plate.
8. Remove the tube attached to the gel and place into a glass beaker. Begin the flow again until all of the acrylamide has flowed out of the gradient maker. Add ddH2O to each of the gradient maker wells and continue the flow to wash all remaining acrylamide out of the gradient maker. Allow the Acrylamide in the beaker to polymerize completely (30 to 60 min); then dispose of the hardened Acrylamide appropriately.
9. Carefully layer 3 to 4 mm Isobutanol or Tertiary Amyl Alcohol with a Pasteur pipette on the top of the gel to prevent oxidation of the polyacrylamide (oxidation will inhibit polymerization).
10. Allow gel to set for 30 to 60 min.
11. Remove the organic top layer.
12. Rinse the top of the gels approximately 5 times with ddH2O.
13. Take apart the gel mold and using a single edged razor blade cut off the polymerized gel waste at the bottom of the gel.
14. Either wrap the gel in Saran Wrap and store at 4°C until ready to use or continue on to pour the stacking gel.
C. Pouring the Stacking Gel
1. Rinse the top of gel out well with ddH2O.
2. Use a piece of Whatman 3MM paper to dry the inner sides of the glass plates above the resolving gel thoroughly. Place the gel between the glass plats, put clamps securely on the glass plates and stand the gel up.
3. Prepare gel Stacking Solution (recipe is for 9 ml):
5.4 ml of ddH2O
2.25 ml of 4X Upper Tris Buffer
1.35 ml of Acrylamide:Bis-Acrylamide Solution (30:0.8)
60 μl of 10% APS
4. Add 30 μl of TEMED to the gel Stacking Solution, invert to mix, and immediately pour the stacking gel.
5. Rinse a comb (with the appropriate number of teeth for the samples to be run) with 100% Ethanol and wipe clean.
6. Place the comb between the gel plates about half way in.
7. Pour the stacking gel, and push the comb in until top of the teeth is level with top of the glass plate.
8. Allow the gel to set for approximately 20 min. Check occasionally to make sure it isn't leaking.
D. Setting Up Gel and Preparing Samples
1. Place the gel in the gel electrophoresis apparatus.
2. Pour 1X SDS Running Buffer into the top reservoir and make sure it doesn't leak.
3. Pour 1X SDS Running Buffer into the bottom reservoir.
4. Remove any bubbles along the bottom edge of the gel by loading a syringe and bent needle with 1X SDS Running Buffer and directing a stream of buffer along the bottom of the gel to force the bubbles out.
5. Boil the protein samples in SDS Sample Buffer for approximately 5 min (don't put samples on ice after boiling).
6. Carefully remove comb from gel.
7. Load samples (1 to 10 μl final volume) using a Hamilton microliter syringe. Rinse the syringe 4X between samples.
E. Electrophoresis of the Gel
1. Electrophoresis the gel at 8 to 15 milliamps constant current until the Bromophenol Blue dye front has moved through the stacking gel.
2. Run the gel at 25 to 35 milliamps constant current as the dye front moves through the resolving gel, until the dye is just off the bottom of the gel.
3. Take apart the gel plates.
4. Use the gel for Western blot, Coomassie protein stain, or silver protein stains (see Protocols for Western Blotting, Coomassie Staining Gels, or Silver Staining Polyacrylamide Gels).
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| SDS Running Buffer (1X) |
| Store at room temperature without the DTT
2% (w/v) SDS
10% (v/v) Glycerol
0.1% (w/v) Bromophenol Blue
*Add DTT right before use
100 mM DTT*
50 mM Tris-Cl, pH 6.8
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| 10% APS |
| 10% (w/v) Ammonium Persulfate
Store at 4°C for several weeks (see Hint #2)
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| Lower Tris Buffer (4X) |
| Add ddH2O to 500 ml
90.85 g Tris base
Adjust pH to 8.8 using HCl
Store at 4°C
20 ml of 10% (w/v) SDS
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| Acrylamide:Bis-Acrylamide Solution |
| Filter through a Whatman #1 Filter paper
0.8% (w/v) Bis-Acrylamide (CAUTION! see Hint #4)
Store at 4°C
30% (w/v) Acrylamide (CAUTION! see Hint #4)
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Bis-acrylamide
Tris-Cl
Bromophenol Blue
Tertiary Amyl Alcohol
Isobutanol
SDS
Hydrochloric Acid
Ethanol
Glycerol
Ammonium Persulfate
Acrylamide
Tris Base
DTT
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1. Bisacrylamide forms cross-links in the Acrylamide gel. The APS drives the polymerization of Acrylamide and Bisacrylamide through the production of free radicals. TEMED catalyzes the formation of free radicals from APS and so speeds the polymerization process.
2. The percentage of Acrylamide:Bis-Acrylamide can vary tremendously. This general protocol describes how to prepare a 7.5% to 15% SDS-Acrylamide:Bis-Acrylamide gel gradient. You can change the low and high percentages as needed for your own use.
3. If the gels are not polymerizing or are polymerizing slower than usual, the problem is often that the APS has gone bad. Make up a fresh solution of APS and re-pour the gel.
4. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
5. Read the manufacturer's diagram for the gradient maker and determine which chamber should contain the lower percentage and higher percentage fluids. Usually but not always fluid flows from the pump to the lower percentage chamber to the higher percentage chamber and finally to the exit valve. Use 1X Lower Tris Buffer to prim the pump and set the flow of the pump such that liquid flows from the 1X Lower Tris Buffer tank into the gradient maker and then into the gel casting mold. This way the pump is not contaminated with acrylamide. Verify the flow before loading the acrylamide solutions into the gradient maker.
6. Once TEMED is added to the mixture the acrylamide begins to polymerize.
7. Do not dispense the acrylamide too rapidly because bubbles may appear trapping oxygen in the gel.
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