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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

-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 sad.gif

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...

-beccaf22-

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? blink.gif

-beccaf22-

Hi beccaf22,

Thanks for sharing your experiences smile.gif
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.

-bullfrog-

Hi,

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

Thank you!

Qinhui Song
email: qsong@bidmc.harvard.edu



QUOTE (ChIPster @ Aug 18 2005, 12:20 PM)
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

-qsong-