3 replies to this topic

[Hallenborg]

Hello ChIP'ping experts.

I hope you can provide a newbie as me with golden tips solving me problems with ChIP.

I am trying to ChIP on cofactors with a 10 minutes formaldehyde-fixation step. Is 10 minutes too short if you are looking at factors not bound directly to the DNA. I add the formaldehyde (1% final) directly to the media.

I am incubating antibody, chromatin and beads overnight. Is that too long?

Is it sufficient to add Complete as a protease inhibitor to the chromatin or should I also add other inhibitors?

Thank you for your valuable assistance.

Cheers,
Hallenborg

[KPDE]

Hallenborg, on Oct 19 2009, 04:43 AM, said:

Hello ChIP'ping experts.

I hope you can provide a newbie as me with golden tips solving me problems with ChIP.

I am trying to ChIP on cofactors with a 10 minutes formaldehyde-fixation step. Is 10 minutes too short if you are looking at factors not bound directly to the DNA. I add the formaldehyde (1% final) directly to the media.

I am incubating antibody, chromatin and beads overnight. Is that too long?

Is it sufficient to add Complete as a protease inhibitor to the chromatin or should I also add other inhibitors?

Thank you for your valuable assistance.

Cheers,
Hallenborg

In my experience, if you've gotten ChIP working with a few other antibodies but you can't get it to work for one factor in particular (assuming you're looking at a positive control region where you expect your factor to bind) it's usually because the antibody you're using doesn't work. Are there any other antibodies you can try?

You can try increasing your crosslinking somewhat (we haven't had problems with crosslinking for 15 minutes and 20 minutes works for some antibodies) though I don't know if that will be enough to make a difference.

You can also give this method a look:

Nowak DE, Tian B, Brasier AR. Two-step cross-linking method for identification of NF-kappaB gene network by chromatin immunoprecipitation. Biotechniques. 2005 Nov;39(5):715-25.

The idea is that the first crosslinking step stabilizes protein-protein interactions before the formaldehyde crosslinking. I haven't tried it myself but a few other papers have cited it.

[Hallenborg]

Thank you very much for a valuable advice.

I will give it a shoot

Edited by Hallenborg, 26 October 2009 - 03:17 AM.

[username]

Hallenborg, on Oct 26 2009, 03:16 AM, said:

Thank you very much for a valuable advice.

I will give it a shoot

I did a ChIP with a cofactor and I incubated it for 10 minutes. It worked fine, but I have to say, that I had a tagged cofactor, and a very good antibody...