Hi
my plasmid has GFP into as gene of interest I did transfect cell ( cell line from mouse) using lipofectamine 3000. I did observe the GFP under a microscope but I could not localize where is it? is it inside the membrane or outside I need to localize where is it exactly?
any suggest?
This is difficult to help answer without an example image. Can you post one?
If by inside vs outside the membrane you mean in relation to the plasma membrane (surface/outer cell vs cytoplasmic/inner cell localisation) then fix a sample of your transfected cells and do an immunostaining with an anti-GFP antibody without permeabilising the cells (no detergent). You can use a secondary antibody conjugated with a different compatible fluorophore. Any signal you detect from the secondary antibody co-localised with a GFP signal will mean a cell surface localisation of your tagged protein.
I agree with Nina and Ulla-Maj. Maybe you could also use a quenching agent such as Trypan Blue to quench surface (membrane) fluorescence of live cells?
Next, CLSM should give you the best results in combination with a membrane marker (DiI or Cell Mask etc.). And, you could try to digest the cell surface of unpermeabilized cells with trypsin, removing proteins located on the surface. If the fluorescence vanishes, it is very likely due to enzymatic removal of a protein on the membrane surface. It all depends on what you want to do with the cells afterwards, of course.
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