Preparing Cell Lysates: Difference between revisions

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