bSLSBio LifeSpan BioSciences, Inc. AMPK Phosphorylation Assay Kit (Fluorometric) LS-K268-100 (100 Tests) • Store at -20°CIntroduction The 5-AMP-activated protein kinase (AMPK) is a key sensor of intracellular energy balance. AMPK is activated in response to an increase in the AMP/ATP ratio which can be caused by a number of factors such as muscle contraction, starvation, or hypoxia. AMPK is a heterotrimeric protein complex comprising of a-(63 kDa), B-(38 kDa) and y-(38 kDa) subunits. For each subunit, isoforms have been identified (a-1, a-2, B-1, B-2, y-1, y-2, y-3) which theoretically allow the formation of 12 different proteins. The a-subunit contains a serine/threonine kinase domain and the regulatory subunits contain binding sites for AMP and ATP and for glycogen. AMPK is activated by phosphorylation on Thr-172 within the catalytic domain. AMP binding results in a 2 to 5-fold increase in AMPK activity compared to the basal level. Binding of AMP to the a-subunit causes allosteric activation of the kinase and induces a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr-172. This cell-based ELISA measures phosphorylated AMPK in whole cells and normalizes the signal to the total protein content. The antibody recognizes both a-subunits and, thus, can be used for cells from all tissues (human, mouse, rat). This simple and efficient assay eliminates the need for cell lysate preparation and can be used to study AMPK regulation in short-term and long-term assays. In this assay, cells grown in 96-well plates are fixed and permeabilized in the wells. AMPK phosphorylation (pAMPK) is measured using a fluorescent ELISA followed by total protein measurement in each well. D. Add fluorogenic substrates. Measure fluorescence intensity. _) Key Features • Sensitive. Can measure pAMPK modulation in as little as 500 cells/well. • Simple and convenient. No cell lysis necessary, cells can be cultured for several days. Total protein and pAMPK can be measured in the same sample. Applications • Determination of AMPK phosphorylation status in whole cells. • Evaluation of direct and indirect modulation of AMPK phosphorylation. • Species tested: human, mouse, rat. FOR RESEARCH USE ONLY! Not for use in humans. LifeSpan BioSciences, Inc. • 2401 Fourth Avenue, Suite 900, Seattle, WA 98121 www.LSBio.com • (206) 464-1554 • [email protected]

bSLSBio LifeSpan BioSciences, Inc. AMPK Phosphorylation Assay Kit (Fluorometric) LS-K268-100 (100 Tests) • Store at -20°CComponents Component Materials Not Supplied 37% formaldehyde (Sigma, cat# F8775); 3% H2O2 (Sigma, cat# 323381); black (clear bottom) cell culture 96-well plate (Sigma, cat# CLS3603); plate sealers (Sigma, cat# A5596); deionized or distilled water; pipetting devices; cell culture incubators; centrifuge tubes; fluorescence plate reader capable of reading at Aex/em = 530/585nm and at Aex/em =360/450nm. Storage This kit is shipped on ice. Upon delivery, store all reagents at -20°C....

AMPK Phosphorylation Assay Kit (Fluorometric) LS-K268-100 (100 Tests) • Store at -20°C For suspension cells, prepare 8% formaldehyde by mixing 2.6 mL of 37% formaldehyde and 9.4 mL of 1×Wash buffer. Centrifuge the plate at 500g for 15 min at 4°C and carefully remove as much media as possible without disturbing the cell pellet (repeat this for suspension cells with each wash step below). Fix the cells in each well by adding 100 μL of 8% formaldehyde to cell pellet. Cover the plate and incubate for 20 min at room temperature. Alternatively, the plate containing the fixed cells can be sealed and...

bSLSBio LifeSpan BioSciences, Inc. AMPK Phosphorylation Assay Kit (Fluorometric) LS-K268-100 (100 Tests) • Store at -20°C Calculations Calculate the mean fluorescence intensities for the Sample Blank ("BLK") wells and "SAMPLE" wells. Subtract the mean fluorescence of the Sample Blank wells from the fluorescence value of the Sample well to yield AF values for the phosphorylated AMPK (AFpampk) at 530/585nm and for the total Protein (AFprot) at 360/450nm. AI"pAMPK- l"pAMpK pAMPK Normalized phosphorylated AMPK (pAMPK) is calculated as, where (AFperk / AFprot)o is the control reference value (e.g....