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Elimination of residual protein in cytosol

localization mitochondria RFP aph

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#1 cellthetruth

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Posted 11 December 2012 - 01:50 AM

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

#2 bob1

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Posted 11 December 2012 - 11:37 AM

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

#3 cellthetruth

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Posted 14 December 2012 - 04:50 AM

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.





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