I have performed a ChIP experiment and I have shared DNA ( 200-500 bp) and I checked if I have well immunoprecipitated the transcription factor of interest by Western Blot (and I succed in this). When I have tried to check the quantity and quality of the chromatin IP with the bioanalyzer It seems that I didn't succeed in immunoprecipitating the chromatin. Have someone already experienced this? Any suggestion? It may seems impossible not to have immunoprecipitated anything. (I have already tried ith the same protocol before and it worked so I don't what happens this time).
How do you know you shared your DNA to 200-500 if not detected?
Then the quantity of DNA immunoprecipitated by CHIP is quiet low, so it sounds difficult to see it without any amplification. Furthermore, how did you manage your DNA from IP to analyse it?
I checked by agarose gel before to immunoprecipitate, unfortunately I will have to sequence after the ChIP but I am not able to validate by qPCR because we did not know the transcription factor of interest binding site. The strange thing is that I have already performed with exactly the same protocol and last time I have very nice peaks on the bioanalyzer.
Thank You for your answers
Well, pretty much everything has already been said, but just to add my opinion:
- as said ChIPped DNA can be quite hard to detect, even with BioA High Sensitivity chips (I had the case last week when building ChiP-seq libraries: almost no detectable DNA, but very nice libraries at the end!)...
- so, you can always check your ChIP with qPCR on a known target of your TF (and a zone known / predicted to not bind it as a negative control),
- how did you check the size of your sheared DNA? Was it after de-crosslinking or before? In my experience, it can change quite a lot.
- last thing: how did you precipitate your DNA after ChIP? Trizol, Phenol-Chloroform, columns? Maybe it was that step that was defective and you lost (most of) your DNA?
Good luck for your ChIP
If you used exactly the same protocol with the same antibody before and got more DNA then most likely your experiment did not work as well this time. You do not even have to notice that you made a mistake (or you even did not make any) - but it does happen that experiments do not work :) Just try to repeat the experiment.
Also when ChIPing for transcription factors (of course it demands on the amount of starting material) then the amount of DNA that you can immunoprecipitate is rather low. I have plenty of experiences where I cannot detect any DNA with Qubit HS assay (actual IPs and also IPs with IgG as a negative control), however, I can easily get enough DNA for sequencing after library preparation and a good enrichment after sequencing.
Of course it would be nice to know some positive regions that could be used to verify that the ChIP has worked prior sequencing. If the sites are not known you can try to predict it based on known target genes and DNA motif analysis (if one is known) but my experience suggest it can take a lot of time and effort to find a good region.
I would suggest repeating the experiment and when you feel good about it (can detect some DNA as you did in the first experiment), make a library and do shallow sequencing (multiplex and share run with other samples). Usually with transcription factors with well defined binding sites (narrow peaks) even million reads is enough to see if your ChIP actually worked. You can then always sequence deeper or at least have now regions that you can use as positive controls for the next experiments.
Also, seeing that IP worked with western blot does not always mean that ChIP will also work. Your transcription factor could bind to DNA weakly or transiently, so even if you can pull down the protein you cannot pull down enough DNA. Especially for proteins that do not bind DNA directly double cross linking (EGS, DSG in addition to formaldehyde ) would be a good thing to try.
Good luck!
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