Immunoprecipitation Optimization

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jonathanjacobs
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Immunoprecipitation Optimization

OK, i'm not sure if this is the right forum for this - the choices seem a bit confusing; so if a moderator moves this thread to another forum; THANKS!

Anyway, I am looking for some tips / feedback on how you successfully pull down huge proteins in an IP. I'm working with a 220kd protein that binds many secondary factors. So far, I can western this protein from whole cell lysates without much trouble, but my IPs are aweful. I've tried 3 different antibodies for the same protein thus far, but it's been 4 months now and my IPs continue to show very little of my target protein is getting pulled down.

any tips you might have would be very much appreciated.

mary-annRSA
mary-annRSA's picture
Have you tried diluting the

Have you tried diluting the protein further. Perhaps leave the antibody concetrations at the same as or only slightly lower than for the westerns. Also have a look at the grade of agarose that you are using - it might be worth pruchasing a premade kit.

Guy Sovak
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Hi,

Hi,
What do you meen by aweful, no result or huge background?
I am doing alot of IP and CO-IP the main protein that is my intrest is ~170kDa and its interaction with HSP's.
It is very trick to get good presentble results. Lot of variables to play with starting with the beads amount and comanies (Amersham works better then sigma in my experiment).
Try different ab, combination and concentration. Try more washing.
Different lysis buffer. For my exp. I am using mild lysis bufer.
Try and tell me if it helped.
Guy

jonathanjacobs
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my IPs dont seem to be

my IPs dont seem to be working well. I usually run a supernatent and pellet lanes side by side and... very little of my target protein is ending up in the pulldown, but there is still not much in my supernatent either. I am probably washing it out, but I've tried lowing salt conc without much better results. Someone in my institute suggested using a "cocktail" of antibodies for the pull down (more than one for the same target). I'm trying that now and I'll have my results in a day or two.

Guy Sovak
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I am using also a combination

I am using also a combination of antibodies with different epitopes.
Waiting to hear your results
Guy

R Bishop
R Bishop's picture
You should also try the

You should also try the Solutions Search - Site Search option for your problem. There are quite a few others dealing with IP problems. Also consider some antibodies dont work with native protein. Finally what are you using for the pulldown? Protein A? Protein G? Many of these are isotype specific.

Rb
Moved to protein isolation in protein chemistry

DatUVa
DatUVa's picture
mary-annRSA wrote:Have you

mary-annRSA wrote:

Have you tried diluting the protein further. Perhaps leave the antibody concetrations at the same as or only slightly lower than for the westerns. Also have a look at the grade of agarose that you are using - it might be worth pruchasing a premade kit.

What would be the advantage of diluting your protein further if you're trying to increase the yield? Wouldn't you want to increase the concentration of antigen available, rather than decrease it?