The basic idea being detection using two colors and see colocalization on the membrane. How to see membrane part first without permeabilizing and then intra-cytoplasmic part too with triton may be over the same fixed cells?
You could transfect the cell with a YFP (yellow-fluorescent protein)-tagged membrane marker - and do the colocalization with a CFP (cyan) tagged chimera of your protein of interest.
A freeze fracture with immunolabeling (FRIL) could be a solution - a possibility to label a large area of membrane. (co-staining is also possible)
For transmembrane proteins, for membrane proteins and even for some membrane associated proteins. But you are right Jean-Marc, you need the machinery and someone who is able to work with.
Well EM is there but we don't have easy access to it. I would like to stick to IF, and my concern is that if I am interested in looking at dosage of protein say a mutant and a WT of the same protein , then how best it should be detected to say that mutants are not less on membrane compared to WT..will using two antibody for the same eprotein ..say one mouse raised and one rabbit raised be used and hence two different Alexa can be used as secondary? definitely tagging is a good option but then sometimes fusion proteins donot give the desired localization.
If you have an antibody recognizing extracellular domains of the membrane protein, you can lightly fix cells (4% paraformaldehyde, 10 min) and perform immunostaining as you regular do (skip permeabilization step, that is). Make sure you run a control in parallel, where you use an antibody that only recognizing intracellular proteins. This control should give no signal if cells remain intact.
Doing IHC is not going to give you a quantitative answer to that question.
If this is a membrane protein and you have antibodies towards the extracellular domain, you can treat with fluorescently labeled antibodies and simply view by confocal microscopy. This allows you to look at live cells. Alternatively the eFP suggestion above is a good one. Good luck!
One of problems is that My proteins N and C terminal are intracellular and hence Ab detection moght not be possible as Ab isnt raised against the whole protein but a small peptide. So the possibilities of detecting such a protein without permeabilizing seems less. And I am not aware what other approach let alone EM, can be doable?
for a quantitative co-localization study you would want to choose a FRET pair and do confocal-FRET. if you just want to know the fraction membrane/intracellular you could do side-by-side western blots on cell lysate and isolated membrane (isolated by centrifugation).
Jack, I am performing the membrane blotting by surface biotinylation. FRET if feasible would be a good option. Tanya, I am not aware if a GFP tag will be detected if I donot permeabilize cells?..I am not aware..
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