Hi friends!
I have 2 proteins of interest which both bind DNA; 1 is a nuclear receptor, and the other is a key component of the molecular circadian clock. These have been reported to physically interact, and I would like to do a ChIP-PCR or ChIP-Seq assay to determine regions of DNA where the proteins bind when complexed. A 2-step ChIP seems logical... first cross-link and immunoprecipitate for 1 protein, then precipitate for protein 2 out of what results from the first pull-down. Are there better methods out there that I am unaware of? Suggestions of a better way? As I said, 1 protein is a nuclear receptor... the other actually acts as a repressor of sorts. Any suggestions are welcome and educational! Thank you!
I am thinking one method was the one you mentioned. Immunopurify with antibody specific binding for each of your target and use a whole cell extract for your control target as reference. Theres another method which you can use a bait attached to a fusion protein which will recognize from your solution one of the 2 proteins. I am not an expert but sometimes the prey is being dragged causes non-functionality in the protein in the end.
https://academic.oup.com/bfg/article/14/1/3/2731578
https://www.thermofisher.com/cy/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-protein-protein-interaction-analysis.html
Chip-seq and overlay the peaks in the downstream analysis. Works pretty well.
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