Methods to quantify human GLUT4 translocation to plamsa membrane - (Sep/12/2014 )

Hi

I'm researching a way to quantify translocated human GLUT4 after insulin stimulation. I would like to know what the gold standard method is for this purpose. I've come across several methods including photobleaching, plasma membrane isolation + western blot, flow cytometry, and ectopic expression of fluorophore-tagged GLUT4 plasmid. But I can't figure out which method is considered the most common one, or gold standard. Do you know of any other methods that I didn't mention? Wouldn't the best method, in theory, simply be to use an antibody that targets one of theextracellular sequences of GLUT4 in combination with a secondary fluorescent antibody for microscopic detection? Although I have not come across anyone who uses this method.

Thank you in advance,

Anna

The problem with your (suggested) simplest method is how do you quantify?  How can you be sure that a signal that you see is uniform across the cell, and how much is translocating?

Western blotting on membrane fractions is probably as good as you could get, but even that might be highly variable, so you would need a bit of practise extracting membranes and making sure that you get the same amount out each time, before you could rely on any results.