SDS Polyacrylamide gels: Separation of proteins by molecular weight.

Biorad Minigel volumes-see handout for assembly instructions.

Using Biorad 30% Acrylamide/Bis-acrylamide mix

Resolving gel. 0.375M Tris, pH 8.8

	5%	7.5%	10%	12.5%	15%
H2O	5.68ml	4.85ml	4.04ml	3.35ml	2.34ml
1.5ml Tris HCl pH8.8	2.5ml	2.5ml	2.5ml	2.5ml	2.5ml
10% SDS	100ul	100ul	100ul	100ul	100ul
Acrylamide/Bis 30% stock	1.66ml	2.5ml	3.3ml	4ml	5ml
10% APS (freshly made)	50ul	50ul	50ul	50ul	50ul
TEMED	5ul	5ul	5ul	5ul	5ul
Total monomer	10ml	10ml	10ml	10ml	10ml

0.75mm Spacer 4.2ml resolving gel needed to completely fill gel sandwich

Stacking gel. 4% gel, 0.125 M Tris, pH 6.8

Solution A	Solution B (Do not chill)
13.3ml 30%acrylamide/Bis stock	25ml 0.5M TrisHCl pH 6.8
36.7ml water	1ml 10% SDS
	24ml H2O

Mix equal volumes of solution A and B. (1ml + 1ml + 3ul TEMED and 20ul APS)

Running conditions

150V, constant voltage. Usual run time is approximately 45 minutes