Too much Background - (Mar/18/2009 )

Hello,
I am completely new in this forum but so far with reading all your questions and answers it was already very helpful

I am trying to do a ChIP assay on stomach tissue and I am using the SIGMA Imprint ChIP Kit.
This kit contains Protein A coated assay wells in which you can bind your specific antibody.
After Antibody Binding and Washing you just add your sonicated chromatin...

My first question would be: Is anyone else using this kit?

So far I got good positive results, however I have a very high background (unspecific binding) with the IgG Control.

So my main question would be: What can I do to minimize the background in a ChIP reaction?

Use less cells per assay well? I used about 2 Million cells per well...
Use another IgG Control? Any recommendations?
Preclearing of my chromatin? Do I need to do that? The SIGMA protocol does not mention any preclearing steps.
Preblocking of tha assay wells? Should they not be already blocked?

It would be very helpful if someone is out there who can tell me how to reduce my unspecific background.

Cheers

-Stefan-

It could be almost anything from wash stringency to the kit itself. Hard to say unless others have been successful with the same procedure or kit.
It may also be the IgG. We were having a similar problem and then switched from a Santa Cruz IgG to a Jackson one and the non-specific background went way down. Have you tried an alternative IgG or better yet, a specific but not applicable IgG like GFP. Keep in mind that "mouse IgG" or "rabbit IgG" are pools of IgGs and there may be one or more in the pool that recognize an epitope from your complex. A specific antibody for a protein not found in your complex may also help reduce background.

-Dr Teeth-

Stefan on Mar 18 2009, 06:54 AM said:

I don't know the kit very well but I would suggest blocking the wells before adding chromatin. And I would suggest doing the chromatin incubation in the presence of blocking reagents as well. We do a 96 well assay (we do our own homemade version) where we block the wells for 1 hour with 5% BSA and 100µg/ml sheared salmon sperm DNA (tRNA works just as well) in IP buffer. We then incubate the antibody with the wells in the same blocking reagents, followed by incubation of the chromatin in the blocking reagents. We still get background but it is much lower than when we don't pre-block the wells or do the incubations with blocking reagents.

As for the IgG. Just leave it out and use empty wells. Using IgG is just as arbitrary as empty wells if you consider how much variation there is between one sera and another. If any reviewers give you a hard time about not using IgG its usually due to their own ignorance of this fact and they'll usually back down when you point this out.

-KPDE-

i tried the invitrogen Magnify chip kit ..has a different mix of magnetic beads and with really low background.
i used around 200,000 cells per IP. maybe try cutting back on cells?

-epi123-