Preparing a SDS-PAGE Gel

Separating Gel

• For 12 mL final volume (2 gels)
• Combine, adding APS and TEMED last
• Pour into cassette, overlay with the top layer of water saturated butanol

Component	6%	7.5%	10%	12.5%
SDS-PAGE Separating Gel Buffer	3 mL	3 mL	3 mL	3 mL
Acrylamide Solution	2.4 mL	3 mL	4 mL	5 mL
Water	6.6 mL	6 mL	5 mL	4mL
APS (make fresh, 0.1g/mL in water)	200uL	200 uL	200 uL	200 uL
TEMED	30 uL	30 uL	30 uL	30uL

• For a Phos-Tag Gel, add 24 uL 5 mM Phos-Tag and 24 uL 10 mM MnCl

Stacking Gel

• Rinse polymerized separating gel with water
• For 10 mL (2 gels)
• Combine, adding APS and TEMED last
• Pour over separating gel and insert comb

Component
SDS-PAGE Stacking Gel Buffer	2.5 mL
Acrylamide Solution	1.7 mL
Water	4 mL
APS (make fresh, 0.1g/mL in water)	200 uL	200 uL	200 uL
TEMED	30 uL	30 uL	30uL