Difference between revisions of "Preparing Cell Lysates"
From Bridges Lab Protocols
Davebridges (Talk | contribs) m (→RIPA Buffer (for 10mL lysis buffer)) |
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===RIPA Buffer (for 10mL lysis buffer)=== | ===RIPA Buffer (for 10mL lysis buffer)=== | ||
| − | {border = "1 | + | {| border="1" |
| − | |-Tris pH7.4 | + | ! || Final Concentration || per 10 mL || Stock |
| − | |-Na Deoxycholate | + | |- |
| − | |-NP-40 | + | |Tris pH7.4 || 50mM || 500uL || 1M |
| − | |-NaCl | + | |- |
| − | |-EDTA | + | |Na Deoxycholate || 0.25% || 250uL || 10% |
| − | |-NaVO3 | + | |- |
| − | |-NaF | + | | NP-40 || 1% || 1mL || 10% |
| − | |-Protease Inhibitors !! 1 tab | + | |- |
| − | } | + | |NaCl || 150mM || 375uL || 4M |
| + | |- | ||
| + | |EDTA || 1mM || 20uL || 0.5M | ||
| + | |- | ||
| + | |NaVO3 || 1mM || 100uL || 1M | ||
| + | |- | ||
| + | |NaF || 5mM || 100uL || 250mM | ||
| + | |- | ||
| + | |NaPPi || 25 mM || 1 mL || 500mM | ||
| + | |- | ||
| + | |- Protease Inhibitors !! 1 tab | ||
| + | |} | ||
==Basic Protocol== | ==Basic Protocol== | ||
Revision as of 21:25, 26 May 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 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
