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Immunoprecipitation without beads?

Immunoprecipitation without beads? - Protocols and Methods Forum

Immunoprecipitation without beads? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 03-10-2005, 01:08 AM
Ben Long
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Default Immunoprecipitation without beads?



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
[Only registered users see links. ]


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  #2  
Old 03-10-2005, 05:27 PM
Stacy
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Default Immunoprecipitation without beads?

In article <422f9df9$[Only registered users see links. ].au>,
"Ben Long" <[Only registered users see links. ].edu.au> wrote:



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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  #3  
Old 03-10-2005, 09:56 PM
Ben Long
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Default Immunoprecipitation without beads?

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" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ]...
sepharose
have
If
do.


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  #4  
Old 03-11-2005, 03:00 AM
D.K.
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Default Immunoprecipitation without beads?

In article <4230c271$[Only registered users see links. ].au>, "Ben Long" <[Only registered users see links. ].edu.au> wrote:

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



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  #5  
Old 03-11-2005, 05:17 AM
Ben Long
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Default Immunoprecipitation without beads?

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." <[Only registered users see links. ]> wrote in message
news:d0r1kf$bqh$[Only registered users see links. ].wisc.edu...
<[Only registered users see links. ].edu.au> wrote:
have
early
an
is
need
care
protein
My
precipitate
I
to
in
to
that
protein
contaminating


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  #6  
Old 03-11-2005, 01:44 PM
D.K.
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Default Immunoprecipitation without beads?

In article <423129f8$[Only registered users see links. ].au>, "Ben Long" <[Only registered users see links. ].edu.au> wrote:

It won't work in your case. The practical limitation of "real" IP
is that you have to have "a lot" (by today's standards) of Ag.

The protocol is exceedingly simple: fix the amount of Ag and
titrate serum amounts to find where insoluble precipitate
forms (increased light-scattering). Too little Ab and each of
its molecule will be bound to 2 Ag molecules. Too much
Ab then each will bind one Ag molecule. Just the right
amount and a network of interactions forms.

DK

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  #7  
Old 03-13-2005, 09:51 PM
Ben Long
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Default Immunoprecipitation without beads?

Many thanks DK. I'll see if I can get anywhere based on your info.

Cheers and best wishes,

Ben

"D.K." <[Only registered users see links. ]> wrote in message
news:d0s7b2$s8j$[Only registered users see links. ].wisc.edu...
<[Only registered users see links. ].edu.au> wrote:


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