Our protein of interest is membrane-bound, and we have been toying with the idea that we may be able to tweak our protocol in order to cut out the permeabilization step and therefore get more specific staining on just the membrane portion. Every protocol I've looked at thus far, however, has required the permeabilization step, and I wanted to know if it's required and/or will affect the experiment in any adverse way if we take it out of the protocol.
Dear Jessica,
Very good question. The short answer is No, you don't have to permeabilise. As Sebastian pointed out, if you have an anti body to the extra cellular epitope of your target protein, it is better not to permeabilise, which can itself introduce its own problems and non-specific unwanted labelling. Once the cells are fixed I think there often enough non-specific large pore formations (may be remnants of hemichannels) on membranes that will allow passing of some antibodies. Sometimes you can get a lot better (more specific and non diffuse) labelling or staining without permeabilising or using 10-fold less detergent than recommended for only 15 minutes. Please also see my following report for similar topics in histochemistry.
best wishes, Refik
Technical Report Research Methods and Technical Considerations: Answers to ov...
I tried this and I didnt get good results.
You can try saponin to permeabilized the cells using a low concentration by short time.
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