Help: Can't see any positive staining at all... SDS PAGE gel - (Oct/30/2006 )

Hi,

I need some help with my SDS-PAGE gel... I can't seem to figure the problem out. Here's what I did:

I ran a 16% gel to determine the molecular weight of a polypeptide I have (polyaspartic acid with some cysteine residues, expected molecular weight ~10kDa). I ran the polypeptide at different concentrations just to make sure overloading wasn't an issue (1mg/mL, 0.1mg/mL, 10ug/mL and 1ug/mL) and ran the gel together with a ladder from bio-rad, and also with bromophenol blue.

I stopped the gel before bromophenol blue reached the end. I did Coomassie staining with one gel but it didn't stain (overnight too). And I did silver staining with another gel, but it also didn't stain (I ran the developer over it a lot of times too). it did stain the ladder though.

What could be the problem? Thanks for the help in advance!

may be gel concentration is not the apropiate one
or
may be there are no proteins in your samples at all, since only the ladder pick up the stain....

Thanks for the reply strawberry.

I'm not really sure about the gel concentration. I've also tried a couple of gel concentrations (10% and 12%) before this but it didn't work either.

Are there any possible issues with typical staining procedures that I may have overlooked? Like staining of hydrophilic/acidic/negatively charged polypeptides?

Maybe there are no proteins in my samples, but I just want to exhaust the possibility of errors I could have made before drawing that conclusion, since I am pretty new to this still.

your peptide seems to have very specific aminoacids. Can they be stained by coomassie blue?
I found that anion of Coomassie Brilliant Blue formed in the acidic staining medium combines with the protonated amino groups of proteins by electrostatic interaction.

so, may be you can't detect by coomassie?
I don't know how silver stain works.

Did you test your peptide concentration? (I am not formilia with peptide.) I think you need at least 100ng peptide to visualize it by coomassie, and about 30ng by silver.. (is that right?)