In vitro AMP kinase assay

In vitro kinase assay was done with recombinant human AMPK α1β1γ1, α2β1γ1, α2β2γ2 and α2β2γ3 isozymes (50 ng per well: SignalChem, Cat# P47-10H-05/P48-10H-05) either with Activator-3 or AMP or AMP and Activator-3 as described in different experiments. A high binding Enzyme Immuno Assay (EIA) 96-well plate (Sigma: Cat# CLS3590) was coated with Acetyl CoA Carboxylase 2 (ACC: 175–271 amino acids) peptide, a direct substrate for AMPK, 100 ng per well (1:1000 ratio, Cat# 12–491, Upstate biotechnology) and incubated overnight at 25 °C. Reaction mixture was incubated at 30 °C for 2 h and reaction was stopped by washing the plate with 0.05% Tween 20 followed by incubation with pACC primary antibody and rabbit secondary antibody as described in cell based ELISA section. Assay buffer is composed of 10 mM DTT, 100 mM magnesium acetate, 50 μM ATP and 10 mM HEPES.