I am working on protein localization in yeast cells. My protein was tagged with GFP. The protein is supposed to be localized at the plasma membrane. But I don't know whether the protein localize at cortical ER or plasma membrane by using fluorescent microscope. Because, the cortical ER and plasma membrane are very closer.
I think that to observe the co-localization of two proteins you should use confocal microscope! It is hard to use fluorescent microscope for this purpose!
Best,
@ Nguyen Nguyen. What I mean that I saw the GFP signal on the boundary of the cell. It might be localize to plasma membrane or to cortical ER. So, How do I know exactly where the protein localize to because the plasma membrane is very close to cortical ER and they are parallel.
I would try ph quenching. gfp is quite sensitive to ph changes and they would occur quickly if the protein is outside but less so in intracellular locations.
get GFP antibodies, get cortical ER antibodies and antibodies for NA/K/ATPase (prevalent plasma membrane protein in most cell types - but check!!) and do double stain with different fluorophores for each antibody. You will need to check antibody compatibility and Immuno-Fluorescence suitability and then titrate your antibodies for every stain. So it is a lengthy process.
If you get greater co-occurence (distinguishment from co-localisation which is positive correlation of intensity as opposed to positive correlation of location which you are more interested in but positive of both is great) of staining for one or the other it is probable that you have an answer. You can also fractionate your samples and see which has greater fluorescence in a spectrofluorimeter.
Definitely use a confocal for your imaging in this instance unless you have access to decent deconvolution software (to make the normal widefield images like a confocal) and take care to image appropriately to allow for deconvolution (take nyquist sampled z-stacks and give a decent overlap at either end in z)
~now for my morning coffee.
Compare figures in the following paper, especially 1A with 1B:
http://www.ncbi.nlm.nih.gov/pubmed/16604528
Depending on how much work you want to put into it, you can also express separate translation products from a single open reading frame using the viral 2A system.
http://www.ncbi.nlm.nih.gov/pubmed/17941043
As others state above, you have to have the appropriate resolution and it helps to have experience about the different membrane compartments in your system.
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