In Article <35210019.27E3 at nospam.ic.ac.uk>, Koen De Smet
<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
>>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?
I used that approach years ago to characterize monoclonal antibodies. We
directly coupled the antibody to the beads, but the protein A/G should work.
The obvious interpretation of your results is that the antibody is not in
fact reacting with the enzyme, i.e. the protein that you think is
responsible for the enzymatic activity is not.
Department of Biological Sciences
University of Alberta
Edmonton, Alberta T6G 2E9
wgallin at gpu.srv.ualberta.ca