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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 (see preparation ) |
100uM |
10uL |
100mM
|
| NaF |
5mM |
100uL |
0.5M
|
| NaPPi |
25 mM |
1 mL |
250mM
|
| Protease Inhibitors |
|
1 mini tab |
|
Basic Protocol
- 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
- Add 200uL Buffer (RIPA buffer) and scrape cells
- Pipet into cold eppendorf tubes
- rotate end over end for 30 minutes at 4oC to lyse
- Centrifuge 10 min at 13,000 RPM to clarify
- Transfer 150uL of lysate to fresh tube and do Bradford assay before add 150uL 2XSDS
- Load gel