KLH vs MAP peptide antibodies

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Sat Jan 2 19:59:49 EST 1999


In article <368eab4f.2898912 at news.uni-hamburg.de>,
behrends at plexus.uke.uni-hamburg.de (Soenke Behrends) wrote:

> Dear Antibody-specialists,
> I desperately need a very good antibody against
> a human cytosolic protein for Immunofluorescence,
> IP and Western. 
> Has anyone tried using the multiple antigenic 
> peptide approach? Did that seem better than 
> coupling the peptide to KLH?  
> Any hints or ideas are appreciated.  
> Thanks a lot for your time
> Soenke

The only time we did a direct comparison between
MAP and KLH-MBS-cysteine-peptide we saw no good
antibody production with the MAP.  We synthesised
the MAP ourselves and had it fully characterised and
the anti-KLH-peptide antibody bound strongly to the
MAP.  The only explanation we could come up with is
that we were using shorter (7mer) peptides than most
people (although I have seen 7mer MAPs used with
good results).  We later immunised the MAP rabbit
with the KLH conjugate and saw a very good response
(actually better than the rabbit with KLH-peptide alone).

The KLH conjugate antibodies work well in westerns
(unpurified) in ELISA (purified) and immunohistochemistry
(purified) and I believe they have also been used
for immunoprecipitation (probably purified).

Purified = IgG by ammonium sulphate and then
depleted of anti-KLH-MBS-cysteine by affinity
chromatography.  Purification using the peptide
doesn't work.

I realise that many people use the MAP with very
good results but I thought it would be best to
warn you that it may not always work whereas we
always saw *something* with a KLH-peptide (even
if it didn't cross-react well with the target
antigen).  I believe it is now routine practice to
include some sort of T-cell response modulator during
immunisation so this may increase the chances of a
MAP working.
     Try both if you have the cash and you are short
on time.  If you only have one shot at it then I would
go for the KLH in the first instance - and then after
you have the grant money rolling in because of your
beautiful blots you can get the MAP synthesised :-)
     Good luck,
          Bernard
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



More information about the Proteins mailing list