i agree - the non-ionic detergent is the most likely
cause of problems and may actually impair solubility
in the presence of SDS
--- Dr Engelbert Buxbaum
<engelbert_buxbaum at hotmail.com> wrote:
> Rafael Garcia wrote:
>> > I am solubilizing difficult proteins using a
> pretty harsh extraction solution:
> > 7M Urea
> > 2M Thiourea
> > 0.05 M DTT
> > 0.01M Tris
> > 2.5% SDS
> > 1% N-lauroylsarcosine (detergent)
> > protease inbitor cocktail
> > pH7.4
> >
> > Something in this mixture makes SDS-PAGE gels look
> streaky and dark.
>> Urea and thiourea can cause problems in SDS-PAGE,
> especially at high
> concentrations. Also the presence of high
> concentrations of non-ionic
> detergent can interfere with SDS-binding to the
> protein.
>> In addition, it might be better to solubilise at the
> pH of SDS-sample
> buffer (6.8) instead of 7.4. You may be interfereing
> with the stacking
> effect of DISK-electrophoresis (see the original
> Ornstein-paper from
> 1962 for a detailed explanation). This too would
> result in broad bands.
>> Are you boiling your sample before electrophoresis?
> This can interfere
> with solubility, try 10 min at 60 degrees instead or
> even, since you are
> working with urea, 30 min at 37 degrees.
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