Immunoprecipitation without beads?

Ben Long long at rsbs.anu.edu.au
Thu Mar 10 16:56:01 EST 2005


Hi Stacy,

Many thanks for your reply.  My goal is to purify a complex for which I have
a polyclonal serum for one of the constituent proteins.  I am in very early
stages of method development at the moment and would be happy to just get an
indication of enrichmnet of the complex using IP.  Nonetheless, the plan is
to eventually characterise the complex using proteomic tehniques, so I need
substantial amounts of the complex some day.  Initially then, I don't care
if there is too much contaminating protein in the final product as I can
develop the method further and perhaps justify the purchase of some protein
A beads and other gear for a more thorough investigation in the future.  My
main question at this moment is can I do IP with just my polyclonal serum
and some free protein A?

Many thanks and best wishes,

Ben

"Stacy" <stacyef at stacyef.net> wrote in message
news:stacyef-C8211C.11274610032005 at news.newsguy.com...
> In article <422f9df9$1 at clarion.carno.net.au>,
>  "Ben Long" <long at rsbs.anu.edu.au> wrote:
>
> > Hi all,
> >
> > I am looking for an immunoprecipitation protocol where I can precipitate
> > antigen/antibody complexes with just protein A (i.e. not bound to
sepharose
> > or agarose beads).  The only reason I want to do this is simply that I
have
> > some protein A available to me at the moment but not protein A beads.
If
> > this can be done and anyone has a protocol I would love to know what to
do.
> >
> > Thanks in advance,
> >
> > Ben
> > ben.long at anu.edu.au
>
>
> What is the goal? Are you trying to purify a protein for subsequent use
> or are you just trying to enrich for a protein (and/or other proteins in
> a muliple protein complex) for subsequent analysis (on a Western, for
> example?)
>
> How pure will your final precipitate need to be? If you've got access to
> 96 well plates optimized for protein binding (e.g. for use in ELISAs)
> then you could try coating some wells with protein A in PBS and use that
> to trap your immune complexes. Alternatively, you could skip the protein
> A and directly bind your antibody to the plate to trap antigen. How I
> would go about doing this depends a great deal on how much contaminating
> protein I'm willing to tolerate at the end so if you post your goal in
> performing the IP in the first place, it would be easier to help you
> design an approach.
>
> Stacy





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