I'm successfully using the SimpleChIP kit from Cell Signaling for ChIP assay. Now I'm trying to establish a protocol for Re-ChIP of two transcription factors, but I'm facing some problems. I only get very low amount of DNA from the second IP and my gene of interest is not or very low amplified in qPCR.
Can someone share a protocol for Re-ChIP or any suggestions?
Thanks in advance!
The first thing what i can recommend you is that use at least 4 times more materials for the first IP. Also start with the antibody which provides better IP percentage in a single IP.
Thanks for your answer!
I already tried to use more chromatin for the first IP, but it wasn't improving. I also do the Re-ChIP in both directions, which means ab1 - ab2 and ab2 - ab1.
Since my protocol and my antibodies are normally working fine in ChIP, I think that the problem is the elution of chromatin-protein-complex after the first IP. I can shortly share my elution protocol:
I'm washing the beads, then I'm adding 2x 60µl 10mM DTT, everytime shaking 30min at 30°C, then collecting the supernatant. After second elution, I dilute 20x to get rid of the high amount of DTT and then using 1ml for the second IP.
I was thinking about using desalting columns instead of diluting the eluate, which should give 20x higher amount of chromatin-protein-complex for the second IP.
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