Preparing Cell Lysates: Difference between revisions
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Materials | ==Materials== | ||
RIPA Buffer (for 10mL lysis buffer) | ===RIPA Buffer (for 10mL lysis buffer)=== | ||
{border = "1" cellspacing = "0" cellpadding = "5" | |||
|-Tris pH7.4 !! 50mM !! 500uL | |||
|-Na Deoxycholate !! 0.25% !! 250uL | |||
|-NP-40 !! 1% !! 1mL | |||
|-NaCl !! 150mM !! 375uL | |||
|-EDTA !! 1mM !! 20uL | |||
|-NaVO3 !! 1mM !! 100uL | |||
|-NaF !! 1mM !! 20uL | |||
|-Protease Inhibitors !! 1 tab | |||
==Basic Protocol== | |||
Basic Protocol | |||
# Stimulate cells if necessary | # Stimulate cells if necessary | ||
# Wash cells 2x1mL with ice cold PBS -/- and aspirate | # Wash cells 2x1mL with ice cold PBS -/- and aspirate | ||
Revision as of 21:14, 26 May 2009
Materials
RIPA Buffer (for 10mL lysis buffer)
{border = "1" cellspacing = "0" cellpadding = "5" |-Tris pH7.4 !! 50mM !! 500uL |-Na Deoxycholate !! 0.25% !! 250uL |-NP-40 !! 1% !! 1mL |-NaCl !! 150mM !! 375uL |-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