Difference between revisions of "Preparing Cell Lysates"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (Created page with 'type something here') |
|||
| Line 1: | Line 1: | ||
| − | + | 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 | ||
Revision as of 15:40, 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
