Koshland Web Site/Methods
SDS-PAGE: gel electrophoresis of proteins

TECHNIQUE is to set up gel plates before you mix the gel mixes. Use the THIN spacers and choose a comb--number of wells varies. Make both resolving gel and stack (NO APS or TEMED)--then add APS and TEMED to resolving gel, mix and pour about 3-3.5ml per gel. Before it polymerises, now add APS and TEMED to your stack mix and pour it gently on top of the resolv. gel (using a pasteur pipette works well). Put in the comb and let solidify. Should take 15-20 minutes. You can keep gels overnight at 4°C if well wrapped to prevent drying out and if you keep the comb in. Note: just before you want to load gels, wash out wells with distilled water to remove unpolymerised acrylamide.

GEL RECEPIES (enough for 2 thin mini-gels)

Percentage of gel 8% 10% 12.5%
30: 0.8% w/v acrylamide:bisacrylamide 2ml 2.5ml 3.1ml
1.0M Tris-Cl pH 8.8 3ml 3ml 3ml
20% SDS 38ul 38ul 38ul
dH2O 2.43ml 1.9ml 1.3ml
Mix together. Add APS and TEMED just before pouring

10% APS 36ul 36ul 36ul
TEMED 5ul 5ul 5ul
7.5ml 7.5ml 7.5ml

STACKING GEL (NOTE pH 6.8)--use 4% stack for <10% res. gel and 6% stack (easier to handle) for >10% resolving gels.

Percentage of stack 4% 6%
30:0.8% w/v acryl:bisacryl 660 ul 1ml
1M Tris-Cl pH6.8 630 ul 630 ul
20% SDS 25 ul 25 ul
dH2O 3.6 ml 3.6 ml
Mix together. Add APSand TEMED just before pouring.

10% APS 25ul 25ul
TEMED 5ul 5ul
5ml 5ml

Preparations of sample: Protein samples are in 1X sample loading buffer (also called Laemmli dye)...ie to 6 ul protein sample, add 3 ul 3X Laemmli dye stock. Boil 3 minutes before loading gel.

Sample loading buffer (Laemmli loading dye) 3X stock:
1M Tris-Cl pH 6.8 2.4 ml
20% SDS 3 ml
Glycerol (100%) 3 ml
B-mercaptoethanol 1.6 ml
Bromophenol blue 0.006g
10 ml (store 4°C)

10X Running buffer (also called Laemmli buffer):
Tris base 30.3 g
Glycine 144 g
SDS 10 g
make to 1L with dH2O

For BioRad apparatus: need 500 ml of 1X buffer so dilute 50 ml 10X stock + 450 ml dH20.

Running gels: Using the BioRad apparatus, run gels at 200V (constant voltage) until the bromophenol blue dye is just off. Takes 50-60 minutes. Gels are run at room temperature.
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