906
edits
Changes
→RIPA Buffer (for 10mL lysis buffer): added link to orthovanadate protocol
|EDTA || 1mM || 20uL || 0.5M
|-
|NaVO3 (see [[Preparation_of_Orthovanadate_Stocks | preparation ]]) || 100uM || 10uL || 100mM
|-
|NaF || 5mM || 100uL || 0.5M
|NaPPi || 25 mM || 1 mL || 250mM
|-
|- Protease Inhibitors !! || || 1 mini tab||
|}
==Basic Protocol==
# Stimulate cells if necessary(i.e. insulin treatment for 10 min)# Wash cells 2x1mL (1mL per well for 12 well plates, 2mL per well for 6 well plates) with ice cold PBS -/- and aspirate# Add 200uL [[Buffer:(RIPA]] buffer ) and scrape cells
# Pipet into cold eppendorf tubes
# rotate end over end for 30 minutes at 4C 4oC to lyse
# Centrifuge 10 min at 13,000 RPM to clarify
# Transfer 150uL of lysate to fresh tube and do Bradford assay before add 150uL 2XSDS
# Load gel