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Star Republic: Guide for Biologists

Protein purification for micro-sequencing --- PVDV

1. Run 1D or 2D protein gel. 3-50 pmole of the protein to be sequenced may be needed. Concentrating the sample before loading if necessary (blowing stream of N2, Speed Vac, or precipitation).

2. Transfer the proteins to the PVDF membrane using a full immersion tank and 10 mM CAPS(3-cyclohexylamino-1-propanesulfonic acid), 10% methanol, pH 11 as the transfering buffer instead of the normal buffer containing Tris and SDS.

3. Staining the proteins on the PVDF membrane with Ponceau S or Amido Black.

4. Cut the desired band.