Anti myc polyclonal antibody

GLUT4 translocation was determined using a colorimetric assay developed by Dr. Amira Klip [36 (link)]. Briefly, L6 expressing GLUT4 tagged with Myc epitope (L6-GLUT4myc) were seeded in 48-well plates and differentiated. Myotubes were serum deprived for 3h prior to experiments, as described above. Half of the wells were treated with 100 nM insulin during the last 20 min of the 3h serum starvation step. Cells were subsequently fixed on ice with 3% paraformaldehyde in PBS for 10 min and washed twice with PBS, before quenching the reaction with 0.1 M glycine for 10 min. After 3 more PBS washes, cells were blocked with 5% goat serum in PBS for 15 min. Myc epitopes were targeted by 1 μg/ml anti-Myc polyclonal antibody (Santa Cruz, #sc-789). Surface-bound Myc epitopes were revealed by exposing myotubes to o-phenylenediamine dihydrochloride reagent (OPD, Sigma, #P9187) for 30 min. OPD is a colorimetric substrate that detects peroxidase activity. Reaction was stopped using 3 M HCl and GLUT4 translocation was quantified by measuring absorbance at 462 nm.