klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Mon Nov 27 21:21:51 EST 2000
In article <F127eJddasH5MrdlZcG0000548e at hotmail.com>, jiesun65 at hotmail.com ("Jie Sun") wrote:
: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
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.
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