colocalization of mitochondrion and GFP fusion - (Dec/17/2007 )

Hi dear brothers and sisters:
I'm writing to consult you an urgent question about the colocalization of mitochondrion and a
transiently expressed GFP-nuclear encoded mitochondrial gene in onion
epidermal cells by particle bombardment.
For several times when I used the 'MitoTrcker Red CMXRos dye' purchased from invitrogene to stain the mitochondrion,
I failed to reach satisfied results：the red signals (should be emitted
from mitochondrion) seemed not overlapped with the green signals, which
should be resulted from the expression of the GFP fusion. Any one who likes to give advices please tell me your email adesses in your reply to this post, so that I can send you some pictures from my experiments. I'd
like you to help me to judge if the MitoTracker stained pattern is normal.
Hoping to hear from you as quickly as possible since I
have only limited time to improve this experiment! Thanks a lot in
advance.
Sincerely yours,
Paul

Well, have you tried using a positive control? Some protein you know is targeted to the mitochondrion for you to check that the dye is working fine.

Moreover, could it be that the GFP-fusion hinders the import of the protein? It's quite a big molecule, maybe somehow it prevents the signaling peptide from being recognized of something like that.

One has to use the right dilution of mitotracker else one can get lots of unspecific staining. You might have to optimise the conc. of mitotracker and confirm it with immunostaining against a mitochondrial antigen.