SDS-PAGE

1. To use commercial BRL boxes: Clean the plates with soap and hot water, wipe dry, scrub with ethanol, wipe dry, and clamp them together with spacers in between. The notched spacers interlock to form a tight seal. I use the thin (0.8 mm) spacers for routine analytical gels, and 1.5 mm spacers to run more sample. I usually use a comb that makes 20 wells.

2. Solutions:

I. 1.5 M Tris-HCl, pH 8.8

II. 1 M Tris-HCl, pH 6.8

III. 30% acrylamide/bisacrylamide (37.5:1). This solution can be bought commercially from Biorad. Store in fridge.

IV. 20% SDS (some people filter)

V. 10% w/v ammonium persulfate, make fresh every month and store in fridge. Note: old bottles of dry AP can go bad.

VI. N,N,N',N'-tetramethylethylene diamine (TEMED). Buy it and store in fridge.

VII. 0.1% SDS. Keep a squirt bottle of it.

VIII. Running buffer: Keep a jug of this. 24 g Tris base, 115.2 g glycine, 20 ml 20% SDS, H2O to 4 liters.

IX. Sample buffer: 100 mM Tris, pH 6.8, 2% SDS, 5% ß- mercaptoethanol, 15% glycerol, enough bromophenol blue to make it dark looking, and optional: 4 M urea. Store in a tightly sealed container to keep the BME from going off. You may still need to add more BME if your solution is old, since it is highly volatile. Some people keep sample buffer -BME and add it right before use.

3. Resolving gel: mix for the correct percentage. A 7% gel resolves down to ~40 kd, and 100 kd runs in the middle of the gel. 10% resolves down to ~20 kd, and 50 kd runs in the middle. Here are recipes for 30 mls, which is enough for two 0.8 mm thick gels, or one 1.5 mm gel.

5% 6% 7% 8% 9% 10%5 6 7 8 9 10 ml 30% acrylamide/bis7.5 7.5 7.5 7.5 7.5 7.5 ml 1.5 M Tris 8.817 16 15 14 13 12 ml H2O

Then add 150 µl 20% SDS400 µl 10% AP20 µl TEMED

Mix gently and pour between plates up to ~1.5 inches below the top, depending on your apparatus. (A rule of thumb: want the stacking gel to be about as deep below the wells as the wells are deep). Immediately and very gently squirt a 0.1% SDS overlay over the acrylamide solution (keeps oxygen away so that the top of the resolving gel polymerizes well). If you squirt this on gently enough you won't disturb the acrylamide solution, and a sharp line of refractive index change will be seen between the gel and the overlay. (Note: a more widely used alternative overlay is water saturated isobutanol. This probably works somewhat better, but it's smelly so I only use it when pouring really thick (3 mm) gels).

4. After the gel polymerizes (10 min) pour off the overlay, and rinse out the well with dH2O. Then pour all the water out, and dry the well by shoving in a paper towel or kimwipes (carefull not to touch the gel!) Insert the comb.

5. Stacking gel : enough for two 0.8 mm thick gels or one 1.5 mm gel

627 µl 30% acryl:bis

780 µl 1M Tris pH 6.8

5 ml H20

31 µl 20% SDS

62 µl 10% AP

3.8 µl TEMED

Pour in between plates, or squirt in with a pasteur pipette. Carefull not to get airbubbles trapped around the teeth of the comb.

6. After polymerization (10-15 min), remove the clamps and bottom spacer.