Difference between revisions of "Preparing Cell Lysates"
From Bridges Lab Protocols
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(→RIPA Buffer (for 10mL lysis buffer)) |
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| Line 5: | Line 5: | ||
! || Final Concentration || per 10 mL || Stock | ! || Final Concentration || per 10 mL || Stock | ||
|- | |- | ||
| − | |Tris pH7.4 || 50mM || 500uL || 1M | + | |Tris pH7.4 || 50mM || 500uL || 1M (pH 8.0@25oC) |
|- | |- | ||
|Na Deoxycholate || 0.25% || 250uL || 10% | |Na Deoxycholate || 0.25% || 250uL || 10% | ||
| Line 15: | Line 15: | ||
|EDTA || 1mM || 20uL || 0.5M | |EDTA || 1mM || 20uL || 0.5M | ||
|- | |- | ||
| − | |NaVO3 || | + | |NaVO3 || 100uM || 10uL || 100mM |
|- | |- | ||
| − | |NaF || 5mM || 100uL || | + | |NaF || 5mM || 100uL || 0.5M |
|- | |- | ||
| − | |NaPPi || 25 mM || 1 mL || | + | |NaPPi || 25 mM || 1 mL || 250mM |
|- | |- | ||
| − | |- Protease Inhibitors !! 1 tab | + | |- Protease Inhibitors !! 1 mini tab |
|} | |} | ||
Revision as of 16:10, 3 November 2009
Materials
RIPA Buffer (for 10mL lysis buffer)
| Final Concentration | per 10 mL | Stock | |
|---|---|---|---|
| Tris pH7.4 | 50mM | 500uL | 1M (pH 8.0@25oC) |
| Na Deoxycholate | 0.25% | 250uL | 10% |
| NP-40 | 1% | 1mL | 10% |
| NaCl | 150mM | 375uL | 4M |
| EDTA | 1mM | 20uL | 0.5M |
| NaVO3 | 100uM | 10uL | 100mM |
| NaF | 5mM | 100uL | 0.5M |
| NaPPi | 25 mM | 1 mL | 250mM |
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
