Variability in ChIP assay - (Aug/18/2005 )

I have been using the EZ-ChIP kit from Upstate. I originally had great success. (i.e. no huge background problems, clear bands, etc.) Now for some reason it has just stopped working even though I am doing the exact same protocol with the same cell type, PCR primers, antibody, etc. Now I am getting high background with my no antibody control or no PCR product.
Has any one else had this experience?
Any suggestions on troubleshooting?
I hate to change my PCR conditions and primers as they were working so well before.
Thanks,
ChIPster

I am also working with an Upstate kit (the original, not EZ) and I seem to get alot of variability as well, in my case, the variability is also in the positive and negative controls so I am eliminiating those results based on "bad technique" I am not really happy with this, it seems like you should always get the same or at least close to the same, but for now I am blaming the complexity of the assay

Does the EZ kit come with a negative control?? How is that reaction, and your positive control behaving? I can say that when I get high background in my negative I see high background in the no Ab samples too...
How do you make the pairs of samples for +/- antibody? I don't like the method that upstate suggests in the kit I have but maybe they have changed it??

Other troubleshooting--are you fixing in media as Upstate suggests? Different batch of media may affect fixation...

It is interesting to find this today, I was going to ask a similar question so I will pose it here:

I did nine seperate fixations, but only three of these were enriched for the positive control and not enriched for the negative control, in all the cases where I get expected results for positive and negative controls my gene is also enriched, but I can't decide if this is okay... is it reasonable to have such variable fixation/IP results?
Is it okay to just throw out results where the positive and negative controls are aberrant or does this say something about my results, like none of them are valid?

Hi beccaf22,

Thanks for sharing your experiences
My experiences is somehow similar with yours: i did three independent ChIP assays, all of this works, but the enrichment fold got variations. Then I repeated once more. in my forth ChIP, I increased the starting material and operated extremely carefully. Therefore, I expected a better result than previous ones. BUT, no enrichment! Even worse, my genes which shown enrichment before gave me a decreased PCR band, which means tubulin control was enriched......so sad

So I asked myself what should I take as real data...And finally I trusted the previous 3 rounds of ChIP and I consider the forth round as "error".

I do feel ChIP is really tricky. If your 3 "successful" ChIP show significant enrichment (at least about 5 fold enrichment), why not feel confidence and take them as real? There are a lot of hidden factors interfere with molecular biology experiments, I have seen ppl run PCR very well on one day but fail on the other day with exactly same reagents and condition.

Hi,

Could you let me know where you buy EZ-ChIP kit ? Cat# and price?

Thank you!

Qinhui Song
email: qsong@bidmc.harvard.edu