Preparing Cell Lysates

Materials

RIPA Buffer (for 10mL lysis buffer)

	Final Concentration	per 10 mL	Stock
Tris pH7.4	50mM	500uL	1M (pH 8.0@25oC)
Na Deoxycholate	0.25%	250uL	10%
NP-40	1%	1mL	10%
NaCl	150mM	375uL	4M
EDTA	1mM	20uL	0.5M
NaVO3	100uM	10uL	100mM
NaF	5mM	100uL	0.5M
NaPPi	25 mM	1 mL	250mM
Protease Inhibitors		1 mini tab

Basic Protocol

1. Stimulate cells if necessary (i.e. insulin treatment for 10 min)
2. Wash cells 2x1mL (1mL per well for 12 well plates, 2mL per well for 6 well plates) with ice cold PBS -/- and aspirate
3. Add 200uL Buffer:RIPA buffer and scrape cells
4. Pipet into cold eppendorf tubes
5. rotate end over end for 30 minutes at 4oC to lyse
6. Centrifuge 10 min at 13,000 RPM to clarify
7. Transfer 150uL of lysate to fresh tube and do Bradford assay before add 150uL 2XSDS
8. Load gel