How to separate vegf dimer into monomer?

Jacob Larsen 91085_at_DFH-Databar at databar.dfh.dk
Mon Mar 3 23:53:52 EST 1997


11 wrote:
> 
> 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
> Biochemistry
> 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.

Cara Baker,
Dept. of Biochemistry,
TCD.



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