high salt IPs - (Jul/10/2006 )
Can high salt conditions (500 milliMolar) be kept throughout the Immunoprecipitation or does it need to be brought dowm to 150 milliMolar after the initial extraction.
I never went higher than 300mM. I think it depends on your protein(s) and the Ab. If the Ab can bind the protein at this molarity of NaCl, then you can keep 500mM through all the IP.
to avoid some protein protein interactions, I already washed my IP with 500 mM NaCl. It works (if your antibody has a good affinity). I have to admit that the signal was weaker.
Sorry for not answering but for asking the forum people 
Why do you extract at high salt conditions? Is the interruption of interactions by high salt conditions allways reversible?
I did once an immunodepletion protocol at 800mM KCl with "home-made" affinity purified Abs and it went fine. But I think that most protein complexes are affected by 400-500mM. Just a small tip: remember to wash once or twice with low salt buffer before boiling your pellets; my first gels ran awfully because of the high salt residue.
Miguelon
Why do you extract at high salt conditions? Is the interruption of interactions by high salt conditions allways reversible?I did once an immunodepletion protocol at 800mM KCl with "home-made" affinity purified Abs and it went fine. But I think that most protein complexes are affected by 400-500mM. Just a small tip: remember to wash once or twice with low salt buffer before boiling your pellets; my first gels ran awfully because of the high salt residue.
Miguelon
i am doing it to extract out nuclear matrix associated proteins. i have tried to dilute the Salt out to 100mM during the IP but upon prolonged incubation in the Ab i observe some clumped up structure which makes the pull down very dirty and ambiguous.
Sorry for not answering but for asking the forum people
Why do you extract at high salt conditions? Is the interruption of interactions by high salt conditions allways reversible?I did once an immunodepletion protocol at 800mM KCl with "home-made" affinity purified Abs and it went fine. But I think that most protein complexes are affected by 400-500mM. Just a small tip: remember to wash once or twice with low salt buffer before boiling your pellets; my first gels ran awfully because of the high salt residue.
Miguelon
i am doing it to extract out nuclear matrix associated proteins. i have tried to dilute the Salt out to 100mM during the IP but upon prolonged incubation in the Ab i observe some clumped up structure which makes the pull down very dirty and ambiguous.
Do you also sonicate? Some people say that if you sonicate the DNA, you can make some of the proteins associated with the nuclear matrix soluble. It worked in my case.
Sorry for not answering but for asking the forum people
Why do you extract at high salt conditions? Is the interruption of interactions by high salt conditions allways reversible?I did once an immunodepletion protocol at 800mM KCl with "home-made" affinity purified Abs and it went fine. But I think that most protein complexes are affected by 400-500mM. Just a small tip: remember to wash once or twice with low salt buffer before boiling your pellets; my first gels ran awfully because of the high salt residue.
Miguelon
i am doing it to extract out nuclear matrix associated proteins. i have tried to dilute the Salt out to 100mM during the IP but upon prolonged incubation in the Ab i observe some clumped up structure which makes the pull down very dirty and ambiguous.
Do you also sonicate? Some people say that if you sonicate the DNA, you can make some of the proteins associated with the nuclear matrix soluble. It worked in my case.
no i hanevnt tried sonication .... just passing it through 21gauge needles but i have huge amounts still left into the pellet that i throw away.