I think one problem here might be that the proteins may have been
crosslinked at several different sites, resulting in conformations that
are not fully denatured by SDS. The presence of differently covalently
crosslinked complexes with the same MW then results in a diffuse band.
Well, at least this is how I interpret my own results...but I really have
not tried to find a way around it. Maybe changing the crosslinker or its
concentration might help?
Actually, even using a crosslinker on a protein that is not crosslinked
to another protein makes the band diffuse, due to the different amounts of
crosslinking agent reacting with each individual protein molecule. A
crosslinked protein molecule population is no more homogenous, that is.
Generally, though, protein bands are sharper on minigels, the running time
being shorter and thus the proteins have less time to diffuse in the gel
during the run.
On 3 Aug 1999, Ossama B Kashlan wrote:
> I'm trying to visualize a crosslinked protein on a polyacrylamide gel, and
> am having some difficulty with sharpness with the heavy bands. My
> expected products have masses between 90 kD and 540 kD. I'm allowing the
> 90 kD monomer to run to the bottom of the gel. I've run a 4% 20 cm x 20
> cm x 1.5 mm gel with a 4% stacking gel. Any suggestions as to how to
> sharpen an expected 540 kD band?
>> Thank you,
> Ossama Kashlan
> University of Pennsylvania
> Department of Chemistry
> Box 233
> 231 S. 34th St.
> Philadelphia, PA 19104
>okashlan at mail.sas.upenn.edu> Lab: (215)898-2927
> Fax: (215)898-2037
>>>
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