How to separate vegf dimer into monomer?
91085_at_DFH-Databar at databar.dfh.dk
Mon Mar 3 23:57:06 EST 1997
> In article <ziping-2802970933350001 at orphan.radonc.washington.edu>,
> ziping at u.washington.edu (Ziping Yang) wrote:
> > Hi, everybody
> > I run vegf western and use B-mercaptoethanol (freshly added to lysis
> > buiffer at 280-400 mM) as a reducing reagent. Samples were boiled before
> > loading to gel. However, VEGF dimer is always there and showed much
> > strong band than monomer did. Any suggestions are wellcomed.
> > ziping
> We have had problems with VEGF dimers and multimers on SDS-PAGE as well.
> The problem seems to be worse with purified protein than with crude
> extracts. We now add 10% (v/v) beta-mercaptoethanol immediately before
> boiling samples and include 1 mM DTT in the resolving gel. This yields
> only monomer on our Westerns. Good luck.
> Fern E. Murdoch
> Uniformed Services University
> Bethesda, MD
How hydrophobic a protein in VEGF? From working with membrane proteins
I have learnt never to boil samples prior to loading on gels. Doing so
results in the formation of apparent aggregates or
'dimers/trimers/tetramers'. So I would try placing samples in sample
buffer ± b-mercapto/DTT at 37 C for 30 min or so before loading and
see if that solves the problem.
Dept. of Biochemistry,
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