2 replies to this topic

[cellthetruth]

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

[bob1]

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

[cellthetruth]

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.