Difference between revisions of "Preparing Cell Lysates"
From Bridges Lab Protocols
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# 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 | ||
| − | # Add 200uL [[Buffer | + | # 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 4C to lyse | # rotate end over end for 30 minutes at 4C to lyse | ||
Revision as of 15:15, 15 July 2009
Materials
RIPA Buffer (for 10mL lysis buffer)
| Final Concentration | per 10 mL | Stock | |
|---|---|---|---|
| Tris pH7.4 | 50mM | 500uL | 1M |
| Na Deoxycholate | 0.25% | 250uL | 10% |
| NP-40 | 1% | 1mL | 10% |
| NaCl | 150mM | 375uL | 4M |
| EDTA | 1mM | 20uL | 0.5M |
| NaVO3 | 1mM | 100uL | 1M |
| NaF | 5mM | 100uL | 250mM |
| NaPPi | 25 mM | 1 mL | 500mM |
Basic Protocol
- Stimulate cells if necessary
- Wash cells 2x1mL 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 to lyse
- Centrifuge 10 min at 13,000 RPM to clarify
- Transfer 150uL of lysate to fresh tube and add 150uL 2XSDS
- Load gel
