General Protocol for Western Blotting - Bio-Rad

Key Solutions and ReagentsLysis buffer: Radioimmunoprecipitation assay buffer (RIPA buffer)50 mM Tris-HCl, pH mM NaCl1% Nonidet P-40 (NP-40) or Triton sodium sodium dodecyl sulphate (SDS)1 mM sodium orthovanadate1 mM NaFProtease inhibitors tablet (Roche)Loading buffer: 2x laemmli buffer4% SDS10% 2-mercaptoethanol20% bromophenol M Tris-HClCheck the pH and adjust to pH if buffer: Tris/Glycine/SDS25 mM Tris190 mM glycine0 .1% S D STransfer buffer25 mM Tris190 mM glycine20% methanolFor proteins larger than 80 kD, we recommend that SDS be included at a final concentration of S staining (w/v) Ponceau S5% glacial acetic acidTris-buffered saline with Tween 20 (TBST) buffer20 mM Tris, pH mM Tween 20 Blocking buffer3% bovine serum albumin (BSA) in TBSTS tripping buffer20 ml 10% ml M Tris HCl, pH ml ultrapure ml 2-mercaptoethanolProcedureSample prep (based on a typical cell culture scenario)

10. Take 20 μg of each sample and add an equal volume of 2x Laemmli sample buffer. 11. Boil each cell lysate in sample buffer at 95°C for 5 min. 12. Centrifuge at 16,000 x g in a microcentrifuge for 1 min. Bulletin 6376 Protocol General Protocol for Western Blotting

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  Samples, Western, Bio rad, Laemmli, Laemmli sample

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