Preparing Cell Lysates: Difference between revisions
→RIPA Buffer (for 10mL lysis buffer): added link to orthovanadate protocol |
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|EDTA || 1mM || 20uL || 0.5M | |EDTA || 1mM || 20uL || 0.5M | ||
|- | |- | ||
|NaVO3 || 100uM || 10uL || 100mM | |NaVO3 (see [[Preparation_of_Orthovanadate_Stocks | preparation ]]) || 100uM || 10uL || 100mM | ||
|- | |- | ||
|NaF || 5mM || 100uL || 0.5M | |NaF || 5mM || 100uL || 0.5M | ||
| Line 21: | Line 21: | ||
|NaPPi || 25 mM || 1 mL || 250mM | |NaPPi || 25 mM || 1 mL || 250mM | ||
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| | |Protease Inhibitors || || 1 mini tab || | ||
|} | |} | ||
==Basic Protocol== | ==Basic Protocol== | ||
# Stimulate cells if necessary (i.e. insulin treatment for 10 min) | # 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 | # 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 | # 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 4oC to lyse | # rotate end over end for 30 minutes at 4oC to lyse | ||