:SDSPAGE.SAM

7.5% POLYACRYLAMIDE GEL

Wear gloves whenever handling polyacrylamide gels

MATERIALS:

Protogel or 30% Acrylamide

Stacking Buffer

Resolving Buffer

Glycerol

TEMED

10% Ammonium persulfate

1.5% Agarose (for large gels)

Laemmli buffer

Molecular weight standards

Alcohol and Kimwipes

Black clips (for large gels)

Glass plates, spacers, combs

Transfer pipets

PAGE running buffer

PRE-EXPT. PROCEDURE:

1. Wash 2 glass plates, 2 spacers (3, for large gel), and 1 comb with glass cleaner. Use alcohol and kimwipes to dry the plates, spacers and comb.

2. For mini-gel: assemble plates and spacers in the clamp of the apparatus

3. For large gel: use black clips to hold the 3 spacers in place around the edges of the glass plates.

4. Seal the sides with 1.5% agarose. Use a black marker and a ruler to draw a line across the glass plate to mark where stacking gel should start.

5. Prepare the two components of the 7.5 % polyacrylamide gel as follows:

Stacking Resolving

Protogel (30% acrylamide) 2 ml 7.5 ml

"Buffer" 7.5 ml 15.0 ml

Glycerol ------- 3.0 ml

Water 5.4 ml 4.2 ml

TEMED 10 ml 20 ml

When you are ready to pour the gel add:

10% ammonium persulfate 150 ml 300 ml

EXPT. PROCEDURE:

1. Use a pipet to pour the resolving gel to the black line. Use a different pipet to pour the stacking gel from the black line to the top edge of the glass plate. Put some stacking gel on the teeth of the comb and insert the comb. Allow gel to polymerize.

2. Once gel has hardened,

For mini-gel: remove the comb and assemble clamps into the electrode assembly.

For large gel: pull off the clips, the bottom spacer and the comb. Use a transfer pipet to place agarose along the top back of the glass plate without ears and insert these plates into the gel apparatus. Hold the plates in place with black clips on both sides. Use a 20 ml syringe with the curved needle to displace any air bubbles which have formed near the bottom edge of the glass plates.

3. Fill the chambers with PAGE running buffer. Flush out the wells using a 20 ml syringe.

4. Prepare enough 2X Laemmli buffer (stock is usually 5x). Put 30 mg/ml of DTT into 2X Laemmli buffer. Add a volume of 2X Laemmli buffer equal to sample volume to each sample.

5. Use a needle to put a hole into the top of each sample tube and then place the tubes in a rack over boiling water. Allow the tubes to boil for 3 minutes.

6. Load your samples into the wells of the gel. Remember to load a molecular weight marker into at least one lane of the gel.

7. For mini-gel: Place the cover on the gel box connecting black to black and red to red. For large gel: Connect the negative (black) electrode to the upper chamber and the positive (red) electrode to the lower chamber.

Adjust the constant current to the desired setting (usually 5 miliamps for an overnight gel) and run the gel for the specified time (mini-gels can be stacked at 50 constant volts and run at 100 constant volts which will take approximately 1.5 hrs.).

BUFFER RECIPES

STACKING GEL BUFFER (1 liter)

20 ml 0.2M EDTA

20 ml 10% SDS

12.1 g Trizma base

add to ~ 900 ml with ddH_2O

adjust pH to 6.7 with H3PO4

bring volume to 1 liter

RESOLVING GEL BUFFER (1 liter)

8 ml 0.5M EDTA

20 ml 10% SDS

90.8 g Trizma base

add to ~ 900 ml with ddH2O

adjust pH to 8.9 with HCl

bring volume to 1 liter

LAEMMLI SAMPLE BUFFER

Prepare 10 ml 2X solution:

0.4 ml 0.1% bromophenol blue

0.2 ml 1M sodium phosphate (pH 7.0)

Prepare 10 ml of 1M NaH PO (monobasic) and 1M Na HPO (dibasic).

Prepare 5 ml of 1M sodium phosphate at pH 7.0 by combining monobasic and

dibasic phosphate solutions while monitoring the pH

2 ml glycerin

4 ml 10% SDS

3.4 ml H_2O

store at room temperature

ELECTROPHORESIS RUNNING BUFFER (16 liter)

97 g Trizma base

456 g glycine

10.7 g EDTA

16 g SDS

dissolve in 4 l of H_2O in large flask, bring up to 16 l in carboy with deionized water.