Cytoplasm and nuclear protein extraction

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Harvest cells and wash with 1XPBS

Cytoplasm extraction

1. Cytoplasm extraction buffer
   • 10mM Hepes or Tris (pH7.5)
   • 40mM KCL
   • 2mMMgCl2
   • 10% glycerol
   • 1mM NaPPi
   • 1ug/mL pepstatin
   • 1ug/mL aprotinin
   • 1ug/mL leupeptin
   • 1mM NaVO4
   • 1mM NaF
   • 1mM PMSF
   • water to 50 mL

Resuspend pellet in 3-5mL of Cytoplasm extraction Buffer. Put into 15mL glass pestle and crush 25-30 times on ice. Spin down at 2000rpm for 5min at 4∞C. Remove supernatant and transfer to microcentrifuge tube. Centrifuge at 14,000rpm for 15minute at 4∞C. Remove supernatant. This is the cytoplasmic protein solution.