EnzyFluo™ AMPK Phosphorylation Assay Kit

Protocol SDS

Application

• For quantitative fluorescent immunoenzymatic assay of AMPK phosphorylation status in cultured cells.

Key Features

• Sensitive. Can measure pAMPK modulation in as little as 500 cells/well.
• New and improved. Total assay time reduced from the standard 21 hours to 6.5 hours (hands-on time 2.5 hrs).
• Simple and convenient. Cells are directly cultured in 96-well plates. No cell lysis is necessary.
• Accurate and high-throughput. Protein phosphorylation is normalized to total cellular protein in the same well, greatly minimizing well-to-well variations. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.

Method

• Cell-Based ELISA (FL530/585 nm, 360/450 nm)

Samples

• Cells

Species

• Human, mouse, rat

Procedure

• Assay takes 6.5 hrs, hands-on time 2.5 hrs

Size

• 100 tests

Shelf Life

• 6 months

More Details

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 α- (63 kDa), β- (38 kDa) and γ- (38 kDa) subunits. For each subunit, isoforms have been identified (α1, α2, β- 1, β- 2, γ1, γ2, γ3) which theoretically allow the formation of 12 different proteins. The α-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. The binding of AMP to the α-subunit causes allosteric activation of the kinase and induces a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr-172.BioAssay Systems cell-based ELISA measures phosphorylated AMPK in whole cells and normalizes the signal to the total protein content. The antibody recognizes both α-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.

Haven’t Found the ELISA Kit for Your Target

We offer two DIY kits: EFC1M-100 for mouse antibodies and EFC2R-100 for rabbit antibodies. All that is required is a primary antibody. If you prefer, we can develop the assay for you. Please email or call 1-510-782-9988 x 2 to request assay service.