SDS PAGE protocol - glasgowlab/home GitHub Wiki

Casting gels

• APS: Make 85ul stocks in Eppendorfs at -80 °C (sensitive to degradation).
• Mix in 50ml Falcon tube. Pour APS and TEMED last, and cast quickly after.

• Cast using syringe and needle. Let set for ~30min. (Check that leftovers are solid)
• Mix in small beaker

• Cast using syringe and needle. Place well comb. Let set for ~30 min.
• Store in Ziploc bag with 1X running buffer, wrap casted gel in paper towel for better wetting. Store at 4°C for up to ~1 week.

Expression and SDS-PAGE

• Spin down 1 ml cells (4000 rpm, 5 min)
• Resuspend in 75 µl BPERII (vortex, 10 min incubate)
• Centrifuge 13,000 rpm / 5 min
• Save supernatant.
  • For running pellet, wash 2x with water
  • Resuspend in 75 µl BPERII
• Add 25 µl of 4x Laemmli buffer.
• Boil at 95°C for 5 min.
• Load 10 µl per lane.