Immunoprecipitation without beads?

Ben Long long at rsbs.anu.edu.au
Fri Mar 11 00:17:43 EST 2005


Many thanks DK,

Do you have one of these "real" IP protocols then?  Perhaps I can IP my
complex with just the polyclonal serum??

Cheers and best wishes,

Ben


"D.K." <dk at no.email.thankstospam.net> wrote in message
news:d0r1kf$bqh$1 at news.doit.wisc.edu...
> In article <4230c271$1 at clarion.carno.net.au>, "Ben Long"
<long at rsbs.anu.edu.au> wrote:
> >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?
>
> No.
>
> The turth is, "immunoprecipitation" in your case is a misnomer. The
> original term referred to a "real" IP where there was no insoluble
> support involved (large aggregates form at the "right" Ab:Ag ratio
> because the Ab is bivalent and polyclonals have more than one
> epitope on the Ag). What today is called IP should be called
> "pull down".
>
> If you are serious about purification then the only straightforward
> and relatively clean way to do it is to immobilize antibody itself and
> do an affinity chromatography over it. Would require at least purifying
> IgG fraction from your sera.
>
> DK
>
>
>
> >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
> >
> >





More information about the Methods mailing list