blurry bands on SDS-PAGE

Emir Khatipov khatipovNO at NOuchicago.edu
Wed Mar 6 13:07:39 EST 2002


Re cysteines: what about boiling with TCEP, a much stronger reducing agent
than BME or DTT?
Emir

"Michael Witty" <mw132 at mole.bio.cam.ac.uk> wrote in message
news:Pine.SGI.4.33.0203061721150.2872327-100000 at mole.bio.cam.ac.uk...
> On Wed, 6 Mar 2002, Richard P. Grant wrote:
>
> > > . . . I think they are caused by residues with pKa's near the pH of
the
> > > running buffer, like Histidine (pKa 6.0) and cysteine (pKa of
> > > thiol/thiolate (pKa about 9).  What do you think?  Regards, Mike.
> > >
> > > (could it be a matter of solubility and caused by Phe and so on?).
> > >
> >
> > First I've heard of it.  Consider what the SDS is doing - it's swamping
> > your protein's charge, so unless you've got something veeeeeeeeery
> > strange . . .
>
>   . . . good point!  Though when I worked on a coiled-coil protein, with 1
> to 3 positive charged residues in every seven it co-migrated with a 50
> rather than the expected 32 kDa marker protein.  Sharp bands though.
> My present protein has a lot of cysteines.
>
> Mike.
>





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