Preparation of PAGE gels.
1. Clean glass plates with ethanol and assemble casting stand, see Biorad instruction manual
2. Mix solutions for lower gel in order shown ie. TEMED last and pour into plates leaving about 2cm at the top. There should be sufficient room to allow such that when the comb is inserted later there is about 5-8mm between the floor of the well and the top of the lower gel.
3. Carefully overlay with DDW and allow to set. Once the gel has set it can be wrapped in gladwrap and stored for several days at least in the fridge.
4. To continue remove overlay water, mix solutions for upper gel in order shown and overlay lower gel. Insert comb and allow to set for 15-20'. Generally the upper gel should not be prepared until the samples are ready as there is a pH difference between the two gels which will diffuse with time.
5. Assemble running unit, see Biorad instruction manual and aspirate bubbles and liquid from wells. Do this immediately before samples are ready as the wells will dry out quite quickly.
6. Load 10ul - 25ul (10ul for a 15 tooth mini comb) and then overlay samples with 1 x SDS running buffer. Empty wells should have 10-15ul sample buffer added and be overlayed as with the test samples so the gel runs straight. Flood the upper chamber by carefully adding 1 x SDS running buffer to unit. Avoid pouring the buffer directly onto the wells.
7. Run gels 200V for 30-45 min until dye front is at the bottom of gel. (Large BioRad gels -15 X 15cm - are run at 35mA per gel).
8. See below for Western transfers. For gels to be stained, stain for 20 min with Coomassie blue solution (see below), then destain in strong destain (50% methanol, 10% acetic acid) 20 min and leave in 10% acetic acid.
Reagents for SDS PAGE
Stock solutions:
A. Acrylamide=30 g acrylamide/0.8 g Bis to 100 ml with Super Q water and filtered through 0.2 µm filter.
B. 1.5 M Tris, pH 8.8= 36.3 g Tris in 100 ml water. pH to 8.8 and adjust to 200ml.
C. 0.5 M Tris, pH 6.8=6 g Tris in 40 ml water. pH to 6.8 and then adjust to 100 ml.
D. 10% ammonium persulfate=0.1 g in 1 ml water
E. 10% SDS
3% Stacking layer: 6.3 ml water/2.5 ml soln C/0.1 ml 10% SDS/1.2 ml soln A/10 µl TEMED/100 µl persulfate
5X Tray Buffer/liter: 15 g Tris/72 g glycine/5 g SDS. Dilute 1:5 for upper tray and 1:10 for lower tray.
