Preparing Cell Lysates

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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

  1. Stimulate cells if necessary (i.e. insulin treatment for 10 min)
  2. Wash cells 2x1mL (1mL per well for 12 well plates, 2mL per well for 6 well plates) with ice cold PBS -/- and aspirate
  3. Add 200uL Buffer (RIPA buffer) and scrape cells
  4. Pipet into cold eppendorf tubes
  5. rotate end over end for 30 minutes at 4oC to lyse
  6. Centrifuge 10 min at 13,000 RPM to clarify
  7. Transfer 150uL of lysate to fresh tube and do Bradford assay before add 150uL 2XSDS
  8. Load gel