Difference between revisions of "Preparing Cell Lysates"

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(edited phosphatase inhibitors)
m (Basic Protocol)
Line 27: Line 27:
 
# 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
# Add 200uL RIPA buffer and scrape cells
+
# Add 200uL [[Buffer/RIPA|RIPA]] buffer and scrape cells
 
# Pipet into cold eppendorf tubes
 
# Pipet into cold eppendorf tubes
 
# rotate end over end for 30 minutes at 4C to lyse
 
# rotate end over end for 30 minutes at 4C to lyse

Revision as of 00:32, 27 May 2009

Materials

RIPA Buffer (for 10mL lysis buffer)

Final Concentration per 10 mL Stock
Tris pH7.4 50mM 500uL 1M
Na Deoxycholate 0.25% 250uL 10%
NP-40 1% 1mL 10%
NaCl 150mM 375uL 4M
EDTA 1mM 20uL 0.5M
NaVO3 1mM 100uL 1M
NaF 5mM 100uL 250mM
NaPPi 25 mM 1 mL 500mM

Basic Protocol

  1. Stimulate cells if necessary
  2. Wash cells 2x1mL with ice cold PBS -/- and aspirate
  3. Add 200uL RIPA buffer and scrape cells
  4. Pipet into cold eppendorf tubes
  5. rotate end over end for 30 minutes at 4C to lyse
  6. Centrifuge 10 min at 13,000 RPM to clarify
  7. Transfer 150uL of lysate to fresh tube and add 150uL 2XSDS
  8. Load gel