Difference between revisions of "Preparing Cell Lysates"
From Bridges Lab Protocols
| Line 2: | Line 2: | ||
RIPA Buffer (for 10mL lysis buffer) | RIPA Buffer (for 10mL lysis buffer) | ||
| + | |||
Tris pH7.4 50mM 500uL | Tris pH7.4 50mM 500uL | ||
| + | |||
Na Deoxycholate 0.25% 250uL | Na Deoxycholate 0.25% 250uL | ||
| + | |||
NP-40 1% 1mL | NP-40 1% 1mL | ||
| + | |||
NaCl 150mM 371uL | NaCl 150mM 371uL | ||
| + | |||
EDTA 1mM 20uL | EDTA 1mM 20uL | ||
| + | |||
NaVO3 1mM 100uL | NaVO3 1mM 100uL | ||
| + | |||
NaF 1mM 20uL | NaF 1mM 20uL | ||
| + | |||
Protease Inhibitors 1 tab | Protease Inhibitors 1 tab | ||
Revision as of 15:42, 11 May 2009
Materials
RIPA Buffer (for 10mL lysis buffer)
Tris pH7.4 50mM 500uL
Na Deoxycholate 0.25% 250uL
NP-40 1% 1mL
NaCl 150mM 371uL
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
