Dear All,
I have been trying to do ChIP qPCRs in Drosophila S2 cells to analyze the recruitment of a protein on certain genes. The problems that I am facing are- the yield is very low (0.001-0.005% of input); When I run the quality control samples after sonification on 1% agarose gels, I see a faint smear, Picture attached (sonification is done 2 times - 9 Cycles on & off, with 30 sec off using Bioruptor). Finally, my ChIP results are somewhat inconsistent, the intergenic regions which I know should not show the recruitment of my protein also show the recruitment after treatment. So, a brief on experiment- Before treatment there should be very little of my protein on target genes and after treatment this should increase significantly. I do see a little increment after treatment but same is happening in intergenic region. Also, my IgG control shows the same.
I would appreciate your help in trouble shooting.
Thank you
Ikram
Ikram what is the protocol are you using for your experiment? On the abcam website you can find either for dynabeads of for simple agarose beads suitable protocol. 2 times 9 cycles for me too much for S2 cells. Your fake result could come form the improper sonication and due to this problem it could be that your low input ratio based on the same problem (simply do not over-sonicate the sample).
BTW i do not see the gel photo...
Dear Tibor
Thank you very much for your reply. I am following the conventional protocol which is the long one, lasting 3 days. Here, I pre-clear the chromatin with protein A beads and incubate for IP overnight at 4`C. I am using Protein A beads for IP. I wash the beads 3 times with low salt buffer, 3 times with high salt buffer, once with LiCl buffer and once with TE buffer before elution.
I apologize that I forgot to attach the photos of the chromatin. I have now attached the photos of 2 different batches of chromatin prepared. The one with faint bands gave me very inconsistent results while the one with bright bands gave me very very low yields (nearly 0.0005-0.0045 % of input) which could all be background (and this has been happening for quite some time now).
I would appreciate your further help.
Thank you
Ikram
First of all the upper gel photo shows something really not good. Than chromatin cold not be used. The lower one seems better, but the sample was reverse crosslinked or not? (i mean what you loaded onto the gel)
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