Grow cells
Harvest
Spin down
Wash with PBS
Spin down enough cells to collect a 50-100 µL cell pellet in a FastPrep tube
Prepare lysis buffer (beforehand) and chill on ice
PMSF: 1 mL per 100 mL buffer
PLAAC: 100 µL per 100 mL buffer
0.5 M Benzamidine: 260 µL per 100 mL buffer
Use fresh PMSF: dissolve 0.035g per 1mL 100%EtOH
2.5 mM MgCl2
3 mM KCl
0.5% Triton X-100
in PBS
PBSMT:
Add protease inhibitors
Add 50-100 µL of cold lysis buffer to cell pellet and vortex
Add 1-2 scoops glass beads (bead level should be just below liquid level)
Mix everything together by vortex
Lyse in FastPrep bead beater
6.5 m/s
40 second lysis
Add 500 µL lysis buffer (keep everything on ice!)
Spin down at maximum speed in tabletop centrifuge for 10 min. (in cold room)
Remove supernatant (lysate) to another tube.