Transcription of General Protocol for Western Blotting - Bio-Rad …
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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 alb
Imaging and data analysis 1. Apply the chemiluminescent substrate to the blot according to the manufacturer’s recommendation. 2. Capture the chemiluminescent signals using a CCD camera-based imager. Note: The use of film is not recommended in this step because of its limited dynamic range. 3. Use image analysis software to read the band intensity
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