Preparing Cell Lysates: Difference between revisions
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==Basic Protocol== | ==Basic Protocol== | ||
# Stimulate cells if necessary | # Stimulate cells if necessary (i.e. insulin treatment for 10 min) | ||
# Wash cells 2x1mL with ice cold PBS -/- and aspirate | # 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 | # Add 200uL [[Buffer:RIPA]] buffer and scrape cells | ||
# Pipet into cold eppendorf tubes | # Pipet into cold eppendorf tubes | ||
# rotate end over end for 30 minutes at | # rotate end over end for 30 minutes at 4oC to lyse | ||
# Centrifuge 10 min at 13,000 RPM to clarify | # Centrifuge 10 min at 13,000 RPM to clarify | ||
# Transfer 150uL of lysate to fresh tube and add 150uL 2XSDS | # Transfer 150uL of lysate to fresh tube and do Bradford assay before add 150uL 2XSDS | ||
# Load gel | # Load gel | ||