Reagents

 

4X TrisCl/SDS pH 8.8 Buffer

  • 91 g Tris base

  • 300 mL H2O

  • Adjust to pH 8.8 with HCl

  • Add H2O to 500 mL total volume

  • 2 g SDS

4x TrisCl/SDS pH 6.8 Buffer

  • 6.05 g Tris base

0.4 g SDS

  • 40 mL H2O

  • Adjust to pH 6.8 with HCl

  • Add H2O to 100 mL total volume

30% acrylamide/0.8% bisacrylamide

  • Store at 4°C in the dark

  • 30.0 g acrylamide

  • 0.8 g N, N'-methylenebisacrylamide

  • Add H2O to 100 mL total volume

5x SDS Electrophoresis Buffer

  • 15.1 g Tris base

  • 72.0 g glycine

  • 5.0 g SDS

  • Add H2O to 1000 mL total volume

Isopropanol Fixing Solution

  • 25% (vol/vol) isopropanol

  • 10% (vol/vol) acetic acid

Rapid Coomassie Staining Solution

  • 10% (vol/vol) acetic acid

  • 0.006% (wt/vol) Coomassie G-250

Destaining Wash Solution

  • 10% (vol/vol) acetic acid


Protocol:

  1. Make fresh 10% Ammonium Persulfate.

  2. Assemble the gel casting apparatus, making sure that the sandwich of glass plates and spacers will make a good seal.

  3. Prepare the Separating Gel solution according to the acrylamide concentration needed.  Vortex.

Separating Gel

 

Final acrylamide conc

5%

6%

7%

8%

9%

10%

12%

13%

15%

30% acryl/0.8% bisacryl

2.5 ml

3.0

3.5

4.0

4.5

5.0

6.0

6.5

7.5

H2O

8.8 ml

8.3

7.8

7.3

6.8

6.3

5.3

4.8

3.8

4x TrisCl/SDS pH 8.8

3.7 ml

3.7

3.7

3.7

3.7

3.7

3.7

3.7

3.7

10% ammonium persulfate

200 µl

200

200

200

200

200

200

200

200

TEMED

10 µl

10

10

10

10

10

10

10

10

  1. Load the apparatus with 4.5 mL of the Separating Gel solution.

  2. Top with ~1 mL of Isoamyl alcohol to isolate the polymerization from oxygen.

  3. After polymerization, pour off the Isoamyl alcohol, and rinse with distilled water.

  4. Remove any water droplets from the inside of the casting apparatus with Whatman paper or a paper towel.  Insert the comb for the stacking gel.

  5. Prepare the Stacking Gel solution. Vortex.

Stacking Gel (5% acrylamide)

H2O

3.0 mL

4x TrisCl/SDS pH 6.8

1.3 mL

30% acrylamide/0.8% bisacrylamide

0.9 mL

10% ammonium persulfate

80 µL

TEMED

5 µL

  1. Load the Stacking Gel solution, taking care not to introduce air bubbles around the comb (some bubbles can be removed by pipetting up and down).

  2. Allow the Stacking Gel to polymerize completely (~45 minutes) before removing comb.

  3. Prepare the samples:

    1. Dilute the protein sample 1:1 with 2x SDS Sample Buffer.

    2. Heat the samples and the molecular weight standards for 5 minutes at 100°C.

  4. Remove the glass and gel sandwich from the casting apparatus.

  5. Clip the sandwich to the electrophoresis apparatus.  Carefully remove the comb from the gel and fill the top of the apparatus with 1x SDS Electrophoresis Buffer.

  6. Using a 20-gauge needle, flush the wells with buffer.

  7. Carefully load the samples into the bottom of the wells using a flat-tipped pipette tip.

  8. Fill the bottom of the electrophoresis apparatus with 1x SDS Electrophoresis Buffer and connect the apparatus to the power supply.

  9. Run the gel at 10 mA until the dye enters the separating gel. Then increase the current to 15 mA.

  10. When the dye reaches the bottom of the separating gel, turn off the power supply, and remove the gel sandwich.

  11. Carefully open the sandwich by using one of the spacers to pry the plates apart.

  12. Gently cut away the stacking gel and place the separating gel in a small plastic container for staining.

  13. Cover the gel with fixing solution and shake gently for 15 minutes.

  14. Pour off the fixer and cover the gel with staining solution. Shake gently for at least 2 hours.

  15. Pour off the staining solution and cover the gel with the wash solution. Destain for at least 2 hours.  (It is usually necessary to change the wash solution at least once)

  16. The gel can be stored in water or dried down between sheets of cellulose on a drying frame.

 

Angela McKay, 22 December 1999
Department of Microbiology
School of Medicine
University of Washington
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