Q: remove enzyme with antiserum?
noone at cancer.bham.ac.uk
Tue Mar 31 11:55:15 EST 1998
In article <35210019.27E3 at nospam.ic.ac.uk>, k.desmet at nospam.ic.ac.uk wrote:
> I am trying to remove selectively an enzyme from a crude extract using
> binding to an antiserum. In short, I hope to bind antibodies to the
> enzyme and then remove this by binding to beads coated with protein A
> and G.
> But it doesn't work. My extract still has the same amount of enzyme
> activity. I tried a number of approaches:
> 1/ first add the antiserum to the extract, incubate overnight at 4C,
> then add beads with protein A/G
> 2/ titrate antiserum in the above experiment
> 3/ first bind serum to beads for 4 hours, then wash and incubate with
> extract overnight at 4C.
> Has anybody ever tried to do a similar experiment, have seen this
> published, or is anybody able to give me advise?
Have you done a western (or an activity assay with your precipitate) to
see whether your antibody actually works in an immunoprecipitation? Not
all antibodies recognize the native antigen. If it does IP, maybe the
amount of enzyme bound is negigible in comparison to the amount of enzyme
present in your lysate (then you have to increase the amount of antibody
(i. e. do a titration)). In case of weak binding, check the buffer
conditions for the IP and use a very mild detergent.
By the way, check "Antibodies" by Harlow and Lane for detailed
descriptions of IP's etc.
Just a few thoughts,
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