protein electrophoresis problems

theodorn at medlib.georgetown.edu theodorn at medlib.georgetown.edu
Thu Dec 10 11:49:37 EST 1998


In article <366FAC48.1BB8 at Bioch.unimaas.nl>,
  M.Harmsma at BIOCH.UNIMAAS.NL wrote:
> Dear colleques,
>
> Hopefully you can help me with the following problem:
> I am trying te estimate the molecular weight of an in vitro synthesized
> membrane protein. I am using SDS-PAGE gelelectrophoresis on a 4%-20%
> Tris-trycine gradient gel, but the radioactively labeled protein doesn't
> move into the gel, but remains at the bottom of the sample well. I have
> tried to avoid formation of aggregates by lowering denaturing
> temperature and/or denaturing time (95 degrees, 30 min. or 15 min. or 3
> min./ 56 degrees, 30 min./ 37 degrees 30min.). I have also tried using 4
> M ureum + 10 mM DTT + 2% SDS in sample buffer and gel electrophoresis
> buffer or 10 mM DTT + 2% SDS in sample- and electrophoresis buffer
> (instead of 2% SDS + 10 mM DTT in sample buffer and only 0.2% SDS in
> gelelectrophoresis buffer). Replacement of DTT by 0.7 M
> beta-mercaptoethanol doesn't help either. Can anyone help me to solve
> this problem?
>
> Thanks,
> Marjan Harmsma
> (M.Harmsma at Bioch.unimaas.nl)
>

Is this a protein synthesized by retic. lysate? Keep in mind that the lysate
is something like 100 mg/ml in protein content, and the usual "add so many
microliters of 3x loading buffer" may not have enough SDS to solubilze all
the protein during denaturation. The standard rule of thumb is to have at
least twice the weight of SDS as protein (but more won't hurt).

Nick Theodorakis

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