V5 epitope

T.Williams bid13 at biol.keele.ac.uk
Thu Jun 7 10:50:36 EST 2001


We have cloned our gene into Invitrogen,s pCDNA3.1/V5-His vector and
have transfecetd these into human B-lymphocyte cell lines.  We have
selected a number of drug resistant clones, but we are really struggling
to demonstrate expression of our fusion protein using the anti-V5
antibody (from Invitrogen).  We have PCRed the clones to demonstrate
that the plasmid has been incorporated.  However every time we try to do
a chemiluminescent Western blot, using the antibody at the recommended
1/5000 dilution, we get non-specific binding to all our proteins.  This
background is so bad that we can see no difference between our vector
controls and our experimental clones.  We have tried nitrocellulose and
PVDF membranes, tried a two step detection system and a one step using
HRP conjugated antibody.  We do not want to use radilabels

Has anyone used this antibody successfully?  What concentration do you
use the antibody?

Please reply  to Sheila  on  bia49 at biol.keele.ac.uk





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