I am planning to conduct an experiment to determine whether a protein of interest binds mitochondrial DNA in mouse brain tissue, but have never done chromatin immunoprecipitation so I'm hoping for some clarification. I've looked through several protocols and all are focused on nuclear DNA, but I wasn't sure how much actually needs to be changed to isolate mtDNA specifically? Will a "standard" ChIP protocol also pulldown mtDNA, and if so, would a qPCR for mtDNA-specific genes tell me if my protein is binding mtDNA?
I'm currently just planning a very rough overview of what I will need, but if anyone has a specific protocol that would be greatly appreciated too.Thank you!
people tried this many times - just didn't work. not because your HCO can hardly transfer through the double membrane, but mainly because it is impossible to reproduce all conditions. Shortly, you can't control neither HCO exposure, nor sonication. The amount if nucleoids is too little and the only way to visualize whether you chop them up into the right size pieces (apx 300 bp) is to isolate them from tissues, not from cultured cells. But again, exposure of tissue samples to crosslinker is a pain in the neck.
Some people tried to isolate mitochondrial particles. Also no luck - upon isolation you do not have the same neighbouring composition of mtDNA/proteins as in living cells.
To sum up. I am sure, one can do it single time and even publish something. But it would be impossible to recapitulate such results.
Better to find some immunofluorescence aporoach than trying to apply chip. Sorry for being to skeptical:)
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts