For final concentration of gel ( % T):
	Separating gel (10 ml)				Stack gel	stack gel
					(10 ml)	5 ml
	7%	10%	12,5 %	15%	5%
30% Acrylamide-bis solution 29:1 (A)	2.33	3.33	4.17	5	1.67	0.835
4x Separation buffer	2.5	2.5	2.5	2.5	­
1.5 M Tris/HCl pH 8.8						0
4x Stacking buffer		­	­	­	2.5	1.25
0.5 M Tris/HCl pH 6.8 + phenol red						0
aqua dest.	5	4	3.2	2.4	5.7	2.85
SDS (10 %)	0.1	0.1	0.1	0.1	0.1	0.05
TEMED	0.015	0.015	0.015	0.015	0.015	0.0075
APS (10%)	0.03	0.03	0.03	0.03	0.03	0.015
total ml	9.98	9.98	10.02	10.05	10.02
Acryl in (A)	0.699	0.999	1.251	1.5	0.501
Acryl %	7.01	10.02	12.49	14.93	5.00
SDS-sample buffer
	final conc	6x		6X Sample Buffer (with DTT)
Tris/HCl pH6.8	0.125	0.75		1 M Tris-Cl (pH 6.8)	2.4 ml
Glycerol	10%	60%		SDS	0.96 g
SDS	2%	12%		Glycerol	4.8 ml
DTT		739	mg	DTT	739 mg	> ca. 600 mM	> final conc. 100mM
Bromophenol blue		4.8	mg	Bromophenol Blue	4.8 mg
					Do not adjust the volume.
Reduction: either by 2% b-Mercaptoethanol
(final conc., equals about 255 mM SH-groups, would mean about 125 mM DTT with 2 SH groups
STRIPPING BUFFER (2% SDS, 62.5mM TRIS pH6.8, 100mM Beta-mercaptoethanol (bME)
Reagent	ml for 500ml	ml for 100 ml
20% SDS	50	10
1M TRIS pH6.8	31.25	6.25
H2O	418.75	83.75
**ADD THE bME FRESH BEFORE USE**
beta-Mercapto	3.55	0.71
Incubate the membrane for 15 - 30 min at 50ºc submerged in stripping buffer.