The peptide I want to see in gel is about 1 kDa, and in the entire amino acid sequence, there are no positively charged residues and only 5 negatively charged residues. I tried to electrophoresis with 12% TRIS-TRICINE gel by dissolving in sample buffer containing SDS in usual way, but the peptide band was not confirmed. I thought that the peptide was small enough to escape from the gel, so after the electrophoresis, it was fixed before the staining but it could not be seen. So I think that peptide size is not a problem, but a problem because the peptide is negative. Is this the right idea? How should i solve it?
Hi Kim,
How are you trying to visualize the peptide band, consider using Silver staining procedure.
You can reverse the electrodes on your gel tank and run the current "backwards" through your gel. Or denature your protein by heating.
The peptide is at the very low end of molecular weight for Tris-tricine gels. I recommend using reverse phase HPLC with ion pairing instead.
Dear Kim,
Your problem is size of the protein and not the charge (As far as SDS-PAGE is concerned). Going for Reverse phase HPLC is the best thing to do. In case if you must have to run SDS-PAGE gel, I would suggest you to try 8-20% gradient gels. Avoid heating of the gel and do fix your proteins as soon the run is over. Stain with silver salts.
Best Wishes
Anil
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