Hi all,

Currently I am doing a protein subcellular localisation by immunofluorescence. The protein should locate on the plasma membrane (it is a fusion protein that does not express naturally). To confirm the protein localisation, I added a V5 tag at the C-terminus of it, which should locate at the intracellular domain of the protein. So I tried the classical immunofluorescence staining, i.e. fixed by 4% PFA then washes, following by Triton-100 permeabilization and BSA blocking before the primary/secondary antibodies staining. The results were not good, since I think the membrane protein was washed out during the permeabilization.

So does someone have tried staining the intracellular domain of a membrane protein? If so, could you tell me which kind of reagent(s) and protocols should be used for the experiment?

Many thanks,

Andy