Elimination of residual protein in cytosol - (Dec/11/2012 )

Hello Scientists,

I would like to target a protein into mitochondria. To do so I fused my protein, an aph3' resistance cassette, to the tagRFP-mito. This RFP contains a mitochondrial targeting sequence, however I have a feeling that not all of the proteins localize to the mitochondria, i.e. there are some left in the cytosol. As I want to check for activity of an antibiotic soleley in mitochondria it is necessary that no aph3' is left in the cytosol. Therefore my question:

Does anybody know how to rapidly degrade specifically proteins in the cytosol?

I have read about some C-terminal degradation through endo-lysosomal pathways. Do you think this would work?

Thank you for reading and giving any advice,

cheers
Martin

You can do mitochondrial preparations by density gradient centrifugation. These should be quite pure and give you the result you want.

Thank you bob1, i thought about this too. But this would give me an in-organello assay instead of a whole-cell treatment. and to check if any residual protein is really left in cytosol the mitochondria can be disrupted (although not all of them) and i would see an artificial band on western. anyway, i'll go for a c-degron and check with confocal microscope for RFP in cytosol. if it worked i'll let you know.