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
