Preparing Cell Lysates

Materials RIPA Buffer (for 10mL lysis buffer) Tris pH7.4 50mM 500uL Na Deoxycholate 0.25% 250uL NP-40 1% 1mL NaCl 150mM 371uL EDTA 1mM 20uL NaVO3 1mM 100uL NaF 1mM 20uL Protease Inhibitors 1 tab

Basic Protocol

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