Harvesting Cell Lysate[Isolation]

Cell Lysis

1. cells plated on a 100mm Petri-dish to sub-confluency
2. pull off media -wash 2x with 7-8 mLs PBS (or HSS media)
3. add 300uL of lysis buffer and spread evenly
4. let stand 10 minutes or so on ice until cells are completely lysed
5. scrape surface of Petri-dish with 1mL syringe stopper or rubber policeman
6. pipette up and down to remove cells from surface of Petri-dish
7. place cells into a 1.5mL falcon tube on ice
8. pull cells through .33 guage syringe several times leave on ice for several minutes
9. spin down, max speed 5-10min
10. place supe in a fresh pre-chilled 1.5mL falcon tube
11. detect protein by western or place in –20 freezer