Hello friends
I am working on Transcription Factor-ChIP on tissue sample, using Diagenode protocol and Kit. I am eluting the DNA using IPure kit (as per the kit recommendations), followed by Qubit quantification for it. I am getting very less concentration (0.047ng/microliter) which I suppose is very less for Sequencing. I wish to know how should I increase the conc of ChIP pull down DNA. I am starting with 0.75 million cells and use magnetic beads method. Also I have put ChIP-qPCR for it, where I am getting a negative results sometime, where ct value of input comes around 26, Ab- 34 and IgG- 35. How should I tackle this?
Maybe you could increase the initating amount of cells and antibody used for ChIP. Based on my own experience, 1-2ng of ChIP-DNA can be gained from 5-10 million cells with 5-10 microgram of antibody.
Hi Pavan
I have checked the cross link timings and the sample is getting sonicated at 50 cycles (30 secs on and 30 secs off ). The other issue is that I am using frozen tissue samples (biopsy material) and the tissue is very limiting. I am using Low cell ChIP kit from diagenode, followed by IPure Kit from the same. I have already standardized my Ab via IHC and western and its coming fine as desired.
I need to ask 1 more thing, if any antibodies immunoexpression is low/ nil, will its ChIP work?
Hi Xuelong Simon Wang
I have very less cells, as I am using frozen tissue samples.
Oh, in that way, you can refer a microChIP (μChIP) protocol, though I have never used before. And as Pavan said, a ChIP-grade (not IHC or WB) antibody is essential to get good IP results.
We have already standardize ChIP protocol for histone studies, so I guess its working. MicroChIP is a good option but will it support NGS?
Moreover, I dont have any ChIP grade antibodies for my transcription factors.
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