Immmunoprecipation question

Jie Sun jiesun65 at hotmail.com
Thu Nov 30 09:42:44 EST 2000


I thought exactly as you said. but I did the control experiment at first 
using one of my standard protein and ab. The protein concentration is 40 
ug/ml, the ab is polycolonal, I dilute 1:10000 for western and works very 
good. I added 1, 3,5, 8, 10, 15 ul antibody(no dilution) to 800 ul my 
standard protein solution and did the immunoprecipation (overnight shaking). 
I used 100 ul beads (from pharmacia) to pull down the complex of antibody 
and protein. I ran SDS-PAGE using the sample from sup and beads. The gel 
showed me almost the same amount protein was pull down although I used 
different concentration antibody. Any suggestions?

Thanks!

Jie




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



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