IUBio

Protein size in the SDSPAGE gel is different from the estimated size

Mitchell Isaacs misaacs at spam_me_not.student.usyd.edu.au
Thu Jun 21 02:34:16 EST 2001


"Hicham Zegzouti" <zegzouti at nature.berkeley.edu> wrote in message
news:f04320400b756e05a9bae@[169.229.61.81]...
> Hi everybody,
>
> I am working on a protein whose estimated MW (from cDNA )is 120KD. I
> overexpressed this protein in E.coli and made an antibody against it.
> Until now normal!
> After purifying the Antibody using Affigel coupled to my protein and
> using this purified Ab aginst transferred plant protein extract, I
> obtain a signal just at around 90 KD. Does anybody have seen
> something like this and does anyone have an explanation for it. What
No problems with that - the theoretical MW often doesn't correspond to the
gel location. Proteins are not as regular as DNA - so don't migrate as
predictably. You could be seeing a fragment - part of the protein may have
been cleaved. Also - have you checked for alternate start positions?

> do you think I can do to check that the band on the western
> corresponds to my protein?
Mass spec, or partial sequencing? Other properties you might know?
(hydrophobicity, absorption spectra).

Cheers,
Mitchell





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