Non-radiometric Cell-free Assay to Measure the Effect of Molecular Chaperones on AMP-activated Kinase Activity.

AMP-activated kinase (AMPK) is a trimeric protein holoenzyme with kinase activity. AMPK plays an important role in cellular metabolism and is thought to function as a fuel sensor within the cell, exerting kinase activity to activate energy-conserving pathways and simultaneously inhibit energy-consuming pathways. Traditional in vitro methods to measure AMPK activity to test potential agonists or antagonists utilize radiolabeled ATP with a peptide substrate. Although radiolabeling provides a high level of sensitivity, this approach is not ideal for medium to high-throughput screening, dose-response curves, or kinetic analyses. Our protocol utilizes Invitrogen's Z'-LYTE™ Kinase Assay Kit (Ser/Thr 23 Peptide) to measure changes in the enzymatic activity of AMPKɑ2β1γ1 in the presence of a molecular chaperone. The Z'-LYTE™ platform is based on Fluorescence Resonance Energy Transfer (FRET). The AMPK peptide substrate (S/T 23 peptide: MRPRKRQGSVRRRV) is a self-contained FRET system, using coumarin as the donor and fluorescein as the acceptor. When the peptide is phosphorylated, it is sensitive to cleavage by a site-specific protease. The cleavage of the phospho-peptide eliminates the FRET pair, and the ratiometric analysis of FRET is used as an indirect measure of AMPK kinase activity. This method does not require the use of radiolabeling or antibodies and is used in a multi-well format, with high reproducibility and throughput capabilities.