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I. ACRYLAMIDE GEL
clean glass plates (windex, ddH20, ETOH) and assemble with spacers, clamps. Adjust to level.
ii) pour gel: 16 cc per BRL vertical gal with 0.8 mm spacer use 37.5:1 acryl:bis, 30% stock (BMB)
of gels 1 1 1 2 2 2
Eacrylamide] 8 10 12.5 8 10 12.5
ddH20 9.65 8.34 6.67 19.30 16.68 13.34
30% Acrylamide 5.36 6.67 8.34 10.72 13.34 16.68
4X LGB Buffer 5.0 5.0 5.0 10.0 10.0 10.0
10% AP * (ul) 66 66 66 132 132 132
TEMED (ul) 10 10 10 20 20 20
make AP f resh q week. Layer butanol (ddH20 sat) on top of acrylamide. Polymerized when third interface apparent (45 min.). Then pour out butanol, wash top of gel with 1X UGB buf f er, and carefully blot dry with Whatman 3MM. Then pour stacking gel (need about 4 ml per gel, although I use 2X f or 1 gel, 4X f or 2 gels) and then place comb into gel:
# of gels 1 2 3 4
ddH20 2.65 5.3 7.95 10.6
30% Acrylamide 0.75 1.5 2.25 3.0
4X UGB Buffer 1.25 2.5 3.75 5.0
68% Glycerol 0.35 0.7 1.05 1.4
10% AP (ul) 15 30 45 60
TEMED 5 10 15 20
This will be ready in - 1 hour.
iii) Remove bottom spacer. Assemble gel apparatus using
clamps. Make up 1 1/2 liters of iX running buffer from the 1OX
stock. Remove comb under buffer. Clean wells by shooting buffer into them using a 19 guage needle. Carefully remove bubbles at the bottom of the gel using a needle bent at 900. I then load 1X running buffer and brief electrophoresis into gel to define the wells. I usually load 30 ug of protein lysate in 30 ul per lane. For each sample, mix 15 ul of 2X loading buffer, 30 ug of lysate, and ddH20 to yield 30 ul (in Eppendorf tube). Heat samples to 100 OC for 3 1/2 to 4 minutes, spin, then load gel. Run at 0.03 Amps at RT for 3 - 4 hours, following dye front and prestained markers. (NOTE: To determine size of band of interest, also run protein size markers, such as comassie blue conjugated proteins (available from BRL, Biorad). These markers have an advantage over biotinylated markers in that you can follow their migration in the gel during electrophoresis and confirm transfer of proteins from the gel to nitrocellulose. The MW are said to be approximate, so other size markers may be needed for more precise size determination.]
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