Preparing Cell Lysates

RIPA Buffer (for 10mL lysis buffer)

{border = "1" cellspacing = "0" cellpadding = "5" |-Tris pH7.4 !! 50mM !! 500uL |-Na Deoxycholate !! 0.25% !! 250uL |-NP-40 !! 1% !! 1mL |-NaCl !! 150mM !! 375uL |-EDTA !! 1mM !! 20uL |-NaVO3 !! 1mM !! 100uL |-NaF !! 1mM !! 20uL |-Protease Inhibitors !! 1 tab }

1. Stimulate cells if necessary
2. Wash cells 2x1mL with ice cold PBS -/- and aspirate
3. Add 200uL RIPA buffer and scrape cells
4. Pipet into cold eppendorf tubes
5. rotate end over end for 30 minutes at 4C to lyse
6. Centrifuge 10 min at 13,000 RPM to clarify
7. Transfer 150uL of lysate to fresh tube and add 150uL 2XSDS
8. Load gel