I did an experiment for a positive control phos-tag system to detect phosphorylated AMPK in min6 cells. I incubated min6 cells for 12h with metformin to induce AMPK phosphorylation then check the result by phospho-specific antibody and get a confirmation of increased phosphorylation but after I check with phostag system I did not get the result that I expected. Anyone has experience in this technique, your feed back is appreciated. here I include my result and condition