Q: remove enzyme with antiserum?

Dr E. Buxbaum EB15 at le.ac.uk
Wed Apr 1 15:21:39 EST 1998


Koen De Smet 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. 

A number of possible explanatios come to mind:

1) Your antibody does not bind to the enzyme. This can happen if it was
raised against denatured protein (ie from SDS-PAGE gels), particularily
with monoclonals. Such antibodies are good for western blots, but not
IPs

2) Your antibody does not bind to Protein A (binding depends on antibody
class and source species)

3) You do not have enough antibody present. Measure affinity and
capacity of your AB prep for the enzyme in question. It is then easy to
calculate the amount of AB necessary for any degree of depletion.

4) Binding conditions are wrong. A composition similar to PBS, at 37
degrees should induce binding in 30-60 min. Once this has been
established, you can vary the conditions to suit your experiment.

5) there may be several isoenzymes present, not all of which react with
the antibody

What I would do in the first instance is to boil the beads after a
binding experiment in SDS-PAGE buffer and do an electrophoresis. You
should see 3 bands on the gel: antibody heavy and light chain and your
enzyme. If you see antibody only, the problem is in antibody-enzyme
interaction, if you don't see the antibody either, its a problem with
Protein A-antibody interactions.

For reasons of cost you may want to consider using Pansorbin
(Calbiochem, formalin fixed Staph. aureus cells) instead of Protein A
agarose, but that is a different matter.



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