Difference between revisions of "Preparing Cell Lysates"
From Bridges Lab Protocols
(→RIPA Buffer (for 10mL lysis buffer)) |
(→Basic Protocol) |
||
| Line 25: | Line 25: | ||
==Basic Protocol== | ==Basic Protocol== | ||
| − | # Stimulate cells if necessary | + | # Stimulate cells if necessary (i.e. insulin treatment for 10 min) |
| − | # Wash cells 2x1mL with ice cold PBS -/- and aspirate | + | # 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 | # Add 200uL [[Buffer:RIPA]] buffer and scrape cells | ||
# Pipet into cold eppendorf tubes | # Pipet into cold eppendorf tubes | ||
| − | # rotate end over end for 30 minutes at | + | # rotate end over end for 30 minutes at 4oC to lyse |
# Centrifuge 10 min at 13,000 RPM to clarify | # Centrifuge 10 min at 13,000 RPM to clarify | ||
| − | # Transfer 150uL of lysate to fresh tube and add 150uL 2XSDS | + | # Transfer 150uL of lysate to fresh tube and do Bradford assay before add 150uL 2XSDS |
# Load gel | # Load gel | ||
Revision as of 16:14, 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 (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
