Difference between revisions of "Preparing Cell Lysates"
From Bridges Lab Protocols
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Davebridges (Talk | contribs) (→RIPA Buffer (for 10mL lysis buffer): added link to orthovanadate protocol) |
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! || Final Concentration || per 10 mL || Stock | ! || Final Concentration || per 10 mL || Stock | ||
|- | |- | ||
| − | |Tris pH7.4 || 50mM || 500uL || 1M | + | |Tris pH7.4 || 50mM || 500uL || 1M (pH 8.0@25oC) |
|- | |- | ||
|Na Deoxycholate || 0.25% || 250uL || 10% | |Na Deoxycholate || 0.25% || 250uL || 10% | ||
| Line 15: | Line 15: | ||
|EDTA || 1mM || 20uL || 0.5M | |EDTA || 1mM || 20uL || 0.5M | ||
|- | |- | ||
| − | |NaVO3 || | + | |NaVO3 (see [[Preparation_of_Orthovanadate_Stocks | preparation ]]) || 100uM || 10uL || 100mM |
|- | |- | ||
| − | |NaF || 5mM || 100uL || | + | |NaF || 5mM || 100uL || 0.5M |
|- | |- | ||
| − | |NaPPi || 25 mM || 1 mL || | + | |NaPPi || 25 mM || 1 mL || 250mM |
|- | |- | ||
| − | | | + | |Protease Inhibitors || || 1 mini tab || |
|} | |} | ||
==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 | + | # 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 | ||
Latest revision as of 20:57, 27 January 2016
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
