Frank O. Fackelmayer
Frank.Fackelmayer at uni-konstanz.de
Wed Jul 5 02:26:37 EST 2000
Johns053 at mc.duke.edu wrote:
> I am looking for protocols for using immunodepletion of crude extracts to
> determine if a protein plays a role in a complex enzymatic reaction. Any
> suggestions and tricks would also be greatly appreciated.
> Thanks in advance,
> Stewart Johnson, Ph.D.
The key to successful immunodepletion is a good antibody. In our
experience, antibodies differ very much in their ability to
immunoprecipitate their antigen. We haven´t been able to find the reason
for this different behaviour, as, eg. antibodies against the same
preparation of antigen but made by different rabbits are not equivalent.
It is best to check a number of different antibodies to thre same
antigen to find one that works well. Interestingly, these are sometimes
not the same antibodies that work well in western blots. You have a fair
chance to find one out of 5 antibodies that IP your protein well, and
these proteins are also the best for immunodepletion.
Some hints for immunodepletion:
* do not use too concentrated extracts for depletion
* if is often necessary to perform two consecutive rounds of depletion
* pre-coupling of antibody to proteinA-Sepharose or paramagnetic beads
(preferred) is sometimes good to reduce the time necessary for depletion
(and thereby preserve enzymatic activities)
Hope this helps,
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