Ampification in no-antibody control of ChIP assay - High background - (Dec/27/2004 )

Hi,

Merry Chrismtas and happy New year.

I am working on Chromatin immunoprecipitation now. I am so upset that my no antibody control also has PCR band. Did anybody meet this kind of problem? What should I do? By the way, I use the ChIP kit from Upstate. I did no template control when I did PCR. There is no band. So it is not because of PCR contamination. Thanks

Difficult to know for sure, especially if you have a good negative control for PCR contamination. My suggestions would be to preclear for a longer period of time, wash for a longer period and possibly do extra washes.

Thanks a lot. I will try.

in addition to prolong the preclear and washing cycles, also be aware of your primer specificity and pcr cycles. there will be some non-specific dna stuck on the beads for sure, but usually PCR using your primers will not amplify it if your primers and pcr program are good. that is why i always use primers with tm around 80 degree. why don't you check your primer Tm and raise the annealing temperature?

I once talked to Upstate technique support about non-specific chipping. Here are their recommendations:

As I had mentioned, you may be experiencing non-specific binding of proteins to the salmon sperm/protein A agarose. To pre-block the agarose, please perform the following:

Incubate/wash the Salmon Sperm DNA Agarose before using it. It can be blocked in 1-5% BSA and Chip dilution buffer to "block" the agarose matrix (you can incubate for 30 minutes mixing at room temperature). After incubation, spin the agarose and remove the 1% BSA/ChIP assay buffer supernantent. Wash once in ChIP assay buffer and continue with the assay as normal.

BTW, I usually get no amplifications in no-antibody controls even without prewahsing.

Hope that helps.

Thanks all of you. I used 1 hour for the preclear step( the kit suggests 30min). Sometimes the no antibody control had no band, sometimes had. The size of the non-specific band is the same as my target fragment. So it still will be there even I change my primers. Maybe I should block the agaose mix first.

Hello everybody!

I am using ChIP assay to study a transcription factor activity in Jurkat cells. I have PCR product in non-specific Ab control. The difference in PCR band intensity between specific Ab sample and control is variable among independent ChIP trials: for the same Abs, many times there is no difference between sample and control in PCR amplification, but sometimes I can get a significant enrichment of PCR product in sample versus control.

I used this method also for IP histones with a protocol from Upstate and everything was OK (same cell line).

The specific Abs used are working well in regular IP.

Does anybody have an optimized protocol for ChIP in case of non-histone proteins and for mammalian cells (eventually lymphocytes)? I am looking for cross-linking and sonication conditions which are efficient for reducing the chromatin non-specific binding to the Sepharose.

Thank you for your help
Val

I have used the ABCAM protocol to great effect. There are prehyb steps for your beads and I always get really clean data using the the standard protocol. Also usually when they say that an Ab is ChIP grade it generally works.

Check it out at: http://www.abcam.com/index.html?pageconfig...rotocol&pid=171

Hi everyone,

Just curious, could you all let me know from what company you buy your proteinA/G agarose from for the ChIP assay? I've tried the salmon sperm DNA/protein A agarose from Upstates, protein A/G agarose from Santa Cruz Biotech, and I still get background signal from my beads (without Ab) pull down. I've also tried blocking the beads with 2% BSA, but the background is still there. I've done water and TE control for my PCR so I know that it is not a PCR problem. So I thought maybe it's the beads. If you're using beads that are working well for you (no background), please let me know. I'd greatly appreciate it.

Thank you