PCR of mitochondrial fraction of nuclear-coded mitochondrial proteins - (Sep/16/2007 )
I am a newby/student and working on proteins that are up-/downregulated in mitochondria after treatment with different drugs.
Anyway, I am looking for a technique to quantify the mitochondrial fraction of different mitochondrial (nuclear-coded) proteins. Since I can't get an ELISA kit for my proteins, I was thinking about quantification of the mRNA.
My problem is that my proteins are not restricted to mitochondria (e.g. Aldolase A - and it's not my housekeeping gene). They also occur in the cytoplasma and/or other compartments. So if I would do a "simple" RT-PCR, I would also measure the fraction of mRNA coding for the proteins occuring in other compartments than the mitochondria.
So, does anybody know about a RT-PCR specific for nuclear-coded mitochondrial proteins?
Thanks in advance!
Have you considered doing the analysis by Western blotting? After all, antibodies for mitochondrial proteins are readily available, not too expensive and it's very straightforward to isolate the cell mitochondrial fraction...
Thanks for the answer, I haven't looked anymore for this entry, bc I thought I would get an E-Mail, if there was an answer, but I haven't.
Anyway, I already did Western Blots, but need a second method for verification!
Well, I do not know much about immunostaining. Anyway, I need two things:
- I must be able to distinguish between
I) isoforms of the same protein being located to mitochondria and
II) isoforms being located to other subcellular locations
- I need to quantify the isoforms in the different subcellular locations (at least in mitochondria)
But about quantifying with this method I do not know????