Immunoprecipitation protocol - Dissociation of protein-g sepharose

Zhonglin Chai Zhonglin.Chai at baker.edu.au
Tue Sep 4 02:34:22 EST 2001


Have you ever tried to cross-link your Ab and ProteinG beads before adding them to your cell lysate for IP? This will dramatically, if not completely, reduce the background of Ig chains in your blot.

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


>>> Melissa Greeve <gliftor at hotmail.com> 09/04/01 03:39pm >>>
Hi There

Like all PhD students, I have come across one of those annoying
'little' hitches that even 5 months of struggling can't seem to solve.

I wonder if anyone can help me. I have been trying to get
immunoprecipitation using protein-G sepharose to work for some time
now, and although I have tried a couple of different buffers, multiple
protocols, slower centrifugation, using unboiled samples and replacing
products, it still won't bring me any joy. Along with the normal
problems of interference from antibody heavy and light chains, due to
the use of the same antibodies for both IP and western blotting, I
have come across a problem that seems to be unique to me....

Has anyone else encountered protein g sepharose dissociating
throughout the IP process??? As you can imagine, this results in a
huge band at 20kD, representing protein G, that binds and fluoresces
when the blot is incubated with any antibody type. This wouldn't be a
problem if I was looking for proteins that don't sit near 20kD, but of
course, 2 of the critical proteins I need to look at are about that
size.

1) Is this normal?
2) How can I stop this happening?? 
3) Any suggestions for perhaps using a more dense acrylamide gel to
separate the bands?
4) Has anyone tried using a non-denaturing acrylamide gel for
immunoprecipitation?
5) Has anyone got a fail-safe protocol for using Sigma protein g
sepharose in IP?

Your help is much appreciated!!!

Cheers,
Melissa




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