906
edits
Changes
→RIPA Buffer (for 10mL lysis buffer): added link to orthovanadate protocol
==Materials==
===RIPA Buffer (for 10mL lysis buffer)==={| border="1"! || 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 371uL|| 375uL || 4M|- |EDTA || 1mM || 20uL|| 0.5M|- |NaVO3 1mM 100uL(see [[Preparation_of_Orthovanadate_Stocks | preparation ]]) || 100uM || 10uL || 100mM|- |NaF 1mM 20uL|| 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