Are there any special protocols I need to follow when blotting onto PVDF membranes?

Yes

1. To reduce the possibility of amino-terminal blockage of the protein:
use the highest purity gel reagents possible

  • make fresh stock solutions for important samples
  • age gel prior to running protein
  • reducing agents should be present during electrophoresis

2.Wear gloves. Handle gels and blots only at the edges to minimize background contamination.

3.The concentration of acrylamide in the lower gel (see B. below) can be from 7-18% but 12.5% is the most useful for 10-70Kd proteins.

4. Use a mini-gel system to maximize protein-to-gel ratio and load as concentrated a protein solution as possible without losing resolution. One wants to load the sequencer with as much protein and as little membrane as possible for best results.

5. Run a set of MW markers on each gel to serve as controls in case the unknown protein is blocked. One can sequence the first few residues of a marker band (ie. lysozyme at 14.3 kD) to make sure the gel reagents didn’t chemically block the protein.

6. If identification of “cys” residues is desired, consider red

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Category: Edman Sequencing

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