I am interested in a protein (say protein X) known to bind to TFAM and mitochondrial DNA. I want to investigate, if this protein is involved in mtDNA transcription and replication. I understand I can knockout the protein and observe its effect. On the other hand overexpression of the protein is also worth looking at. But as I am new in this field, I would appreciate, if anyone can suggest any other techniques, something more specific for the mitochondria.
There are a number of proteins known to interact, as for eg., mitochondrial transcription factor B1 to interact with C-terminal activation region of h-TFAM; but my suggestion would be to go for immunoprecipitation of isolated mitochndrial & nuclear proteins using high titer anti-TFAM antibodies; and identify the co-immunoprecipitated proteins by sequencing. You may end up with one or more that has effect on mito-biogenesis. This may be tested by using DRP1, Opa1, Mfn1 and mfn2 levels in a human cell line that undergoes mitophagy (or cells which undergo mitochondrial biogenesis crisis) as a model system.
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