Preparing a SDS-PAGE Gel

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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 in the separating gel only

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
TEMED 30 uL