Immmunoprecipation question

Martin Offterdinger martin.offterdinger at akh-wien.ac.at
Tue Nov 28 07:23:14 EST 2000


"Dima Klenchin" <klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu> schrieb im
Newsbeitrag news:8vv4rv$2d0_004 at doit.wisc.edu...
> In article <F127eJddasH5MrdlZcG0000548e at hotmail.com>, jiesun65 at hotmail.com
("Jie Sun") wrote:
> :Hi, there,
> :
> :My understanding for immunoprecipation is to pull down trace protein
using
> :excessive antibody. My question is: Is it possible to pull down large
amount
> :of protein (let say 40 ug/ml) by IP? I am wondering how much antibodies
and
> :beads should be used?
>
> Sure. If you have pure Ab of high affinity, then at least
> equimolar or twice that to be on a safer side. Assuming you have
> antiserum, take it that IgG concentration there is ~ 1 mg/ml,
> and only 10% of them will bind your protein. Then use 3-5 molar
> excess from that estimate.
>
> As far as beads go, typical Protein A Sepharose has 5-10 mg
> IgG/ml of settled gel capacity. That theoretically means that tiny
> amounts of "beads" may suffice (~20 ul for pure Ab). In practice the
> amount of Sepharose should not be less then 10% of final volume for
> efficient pull down (in any case, make sure you always add enough
> added to bind all IgG present). Allow at least 1 hr or better o/n in
> cold room.
>
> However, if your antigen is very dilute, doing IP with the aim to
> get 40 ug is simply impractical (too expensive, messy and results
> in very impure prep). You'd be better off purifying IgG on Protein
> A, coupling them covalently to Sepharose and doing standard
> affinity chromatography in column.
>
>         - Dima

I would rather omit large scale IP and use immunoaffinity purification on a
column instead of it like that:

Immobilize Ab on column.
Apply sample
Wash
Elute (eg. low pH buffer)
Neutralize

regards
martin







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