Hi Everyone,
I have some problems with my ChIP which used to work fine on other cell types.
I used HeLa cells for this one. The major problem is that both my 'protein of intersest' IP and 'positive control' IP (where I know that the protein binds to the specific DNA region) gave very strong signals (Ct between 22 and 25) for my positive control region and, unfortunately, for my negative control IgG (similar Ct).
I also included a cell line depleted for my protein of intrest with shRNA as a control and unfortunately, I had sigals amplified for all the IPs, even with the IgG.
Can anyone help me please?
Thank you in advance :)
In addition to a positive control region I would always include a negative control region (which tour TF of interest should not bind). Comparing these two regions should give you a better indication if the IP actually worked than comparison with IgG. I have seen plenty of experiments where IgG gives as high signal as IP but there should be no difference between the positive and negative regions. If you do not see enrichment in your IP then you need to do more troubleshooting. Of course it is possible that the binding sites for your TF of interest are different in different cell types so you can include multiple positive regions. If you do not know any positive regions for sure then low coverage sequencing should give you an idea of the IP quality and give you some positive regions.
Good luck!
It is normal for IgG ChIP to have peaks. We have made IgG ChIP-seq and have seen clear peaks. So it is possible that this point can be false positive. But if you had normal results with this point you can try to change your protocol. For example an increase of number or duration of washes can help with high signal from IgG.
Thank you Tonis and Nikolay for your answers! I will redo the experiment this week with some changes taking into account your advices, I hope it will work!
I'll let you know for the results.
Best!
One simple think you can do is use a different set of primers around the same region.
Sometimes it is the primers that are problematic.
good luck
In order to assess the specificity of a ChIP reaction, it is recommended to analyze in qPCR 1 or 2 regions enriched for the protein of interest and 1 or 2 regions unenriched for the protein of interest. The ratio of the enrichments for the region enriched (positive controls) over the enrichment for the region unenriched (negative regions) will inform you about the specificity of the ChIP assay. Performing a titration of the antibody (using different antibody amounts per IP) may also help to increase the ChIP specificity.
If the ChIP reaction is specific you should not have a strong enrichment in the negative control region but it sometimes happens to have enrichment.
The quantification of the samples is also an indication. You should recover more DNA in the IP with the antibody against the protein of interest compared to the IP with the IgG.
I hope it will help,
https://www.diagenode.com/
Hi Elias,
Where you able to solve the problem with IgG? What are the changes you made to your protocol?
Thank you.
You can increase the washing steps and also perform a preclear step of the sample
if you have positive signal even in the depleted cell line, I don't think the problem is the high signal IgG since the KO cell line should be a perfect negative. Maybe a contamination in your PCR?
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