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This is for up to 2 10cm plates of cells

1) Trypsinize as usual and spin down . Resuspend in 2ml of PBS and split into 2 microfuge tubes.

2) Spin 5 sec in microfuge (full speed) and resuspend in 1 ml of Buffer A.

3) Spin down again for 5 sec and resusupend in 1ml of Buffer A-set on ice for 10 min. to swell and lyse cells.

4) Vortex for 30 sec and check for lysis-if lysed spin for 5 sec and discard sups

5) Resuspend pellet in equal volume of Buffer C and set on ice for 15 min.

6) Spin 5 min at 40C in microfuge and take supernatant--aliquot and freeze at -800C


Buffer A (50 ml)

500ml 1M HEPES, pH 7.9

75ml 1M MgCl2

500ml 1M KCl

25ml 1M DTT

100ml 0.1M PMSF

49 ml dH2O

Buffer C (10 ml)

100ml 1M HEPES, pH 7.9

2.5ml glycerol

840ml 5M NaCl

15ml 1M MgCl2

4ml 0.5M EDTA

5ml 1M DTT

50ml 0.1M PMSF

6.4ml dH2O