Re: Peptides & Coomassie R-250
Dan Crimmins (crimmins@pathbox.wustl.edu)
Mon, 23 Aug 1999 08:17:20 -0500
At 11:11 AM 8/23/1999 +0100, Jenny Shipway wrote:
>
>Hello list,
>
>I've been playing with IEF recently, but when I stain (Coomassie R-250) my
>gels (Phastgel) I can only see the standards. They're present at about
>50ng each, while my peptides (30aa) remain invisible even at 1000ng.
>
>Are there any possible reasons why the dye may not be sticking to my
>peptides? They're not massively exciting. Should form coiled-coils.
>
>Alternatively, do I just need more peptide on there? The book said to use
>between 60 and 2000ng. Considering the visibility of the standards, I'd
>have thought I should have seen something at 1000ng.
>
>Thanks in advance,
>
>Jenny Shipway
>Centre for Biomolecular Design and Drug Development
>University of Sussex
>
>
>
>
Jenny,
Are you sure that your peptides are not diffusing out of the gel during
the staining/destaining process? You may have to try glutaraldehyde
x-linking to anchor the peptides in the gel. If you are using Urea >8M for
sample prep, you may no longer have coiled-coils either.
Regards,
Dan L. Crimmins
Washington University School of Medicine
Dept. Pathology/Division of Laboratory Medicine
660 S. Euclid Ave., Box 8118
St. Louis, MO 63110
Phone: 314-454-8514; Fax: 314-454-5208
e-mail: crimmins@labmed.wustl.edu