Immunoprecipitation

ChemieNieuws niemand at chem.vu.nl
Fri Jul 27 08:18:12 EST 2001


Check if your antibody is recognized by Protein A-agarose (Boil your beads
with LSB and analyse by SDS-PAGE: a heavy and light chain must apear).
Monoclonal mouse is poorly bound by Protein A, as is my experience. You can
use a 'brigde antibody': a rabbit-anti-mouse IgG.

good luck.

Pieter van Breugel
Biochemie en Moleculaire Biologie
Vrije Universiteit
De Boelelaan 1083
1081 HV Amsterdam
Room KB266/Lab KA263
Tel. (+31)20 444.75.49/64/63
Fax: (+31) 20 444.7553

""Zhonglin Chai"" <Zhonglin.Chai at baker.edu.au> wrote in message
news:sb5e94f9.046 at baker.edu.au...
One possible reason is the solubility of the protein as you mentioned in
your message. But it is not clear how possibly you would be able to know it
is a "soluble" nuclear factor by immunocytochemistry labelling. To determine
whether your protein is present in the soluble fraction of the cell lysate,
you may just do a Western using anti-myc antibody to detect your protein in
the supernatant you will use for IP.

Secondly you did not tell us how you detected the precipitated proteins. Did
you label the proteins with 35S, or what? Not all the proteins are labelled
equally well. To determine whether your protein is precipitated you may just
load your precipitated proteins to gel and do another Western. You'd better
have a negative control using isotype control antibody in your IP that
should give you a negative result by Western.

To reduce the IP background, you may need to pre-clean your lysate using
control antibody and beads, or use less antibody, or dilute your lysate, or
incubate for a shorter time, etc.

Hope this of help.

Zhonglin Chai

Zhonglin Chai, PhD

Molecular Hypertension Laboratory
Baker Medical Research Institute
P.O. Box 6492
Melbourne 8008
Victoria, Australia
Telephone: +61 3, 9522 4357
mobile : 0413 58 1940, or international +61, 413 58 1940
Fax: +61 3, 9521 1362
email: zhonglin.chai at baker.edu.au


>>> Natalie Sampson <nsampson at hgmp.mrc.ac.uk> 07/24/01 09:01PM >>>
I am trying (without success) to pull down a transiently expressed c-myc
epitope tagged protein.  From immunocytochemistry labelling, my protein .
>From the precipitation experiments I have done so far, I am getting several
background (unwanted proteins) and not mine.  It is definitely present in
the transfected cells (western blot analysis on total cell lysates).  Does
anyone have any suggestions.  I have tried numerous washing buffers (RIPA,
high salt etc) and am fast running o
ut of ideas.  Can anyone  help?

Thank you

Natalie


nsampson at hgmp.mrc.ac.uk

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