Difference between revisions of "Preparing Cell Lysates"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (edited table) |
|||
| Line 1: | Line 1: | ||
| − | Materials | + | ==Materials== |
| − | RIPA Buffer (for 10mL lysis buffer) | + | ===RIPA Buffer (for 10mL lysis buffer)=== |
| + | {border = "1" cellspacing = "0" cellpadding = "5" | ||
| + | |-Tris pH7.4 !! 50mM !! 500uL | ||
| + | |-Na Deoxycholate !! 0.25% !! 250uL | ||
| + | |-NP-40 !! 1% !! 1mL | ||
| + | |-NaCl !! 150mM !! 375uL | ||
| + | |-EDTA !! 1mM !! 20uL | ||
| + | |-NaVO3 !! 1mM !! 100uL | ||
| + | |-NaF !! 1mM !! 20uL | ||
| + | |-Protease Inhibitors !! 1 tab | ||
| − | + | ==Basic Protocol== | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | Basic Protocol | + | |
# Stimulate cells if necessary | # Stimulate cells if necessary | ||
# Wash cells 2x1mL with ice cold PBS -/- and aspirate | # Wash cells 2x1mL with ice cold PBS -/- and aspirate | ||
Revision as of 21:14, 26 May 2009
Materials
RIPA Buffer (for 10mL lysis buffer)
{border = "1" cellspacing = "0" cellpadding = "5" |-Tris pH7.4 !! 50mM !! 500uL |-Na Deoxycholate !! 0.25% !! 250uL |-NP-40 !! 1% !! 1mL |-NaCl !! 150mM !! 375uL |-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
