The Human AMPK alpha-1,2 (Phospho) [pT172] ELISA Kit is a solid-phase sandwich Enzyme-Linked Immunosorbent Assay (ELISA) designed to detect and quantify the level of AMPK alpha-1,2 (Phospho) [pT172] in fresh or frozen human cell lysates. Cross-reactivity has been observed in mouse and rat cells. The assay recognizes both natural and recombinant human AMPK alpha-1,2 (Phospho) [pT172].
Principle of the method
A monoclonal capture antibody specific for AMPK alpha-1,2 (Phospho) [pT172] has been coated onto the wells of the 96-well plate. During the first incubation, standards of known content and unknown samples are pipetted into the wells and the antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for the target protein is added to the wells and serves as a detection antibody by binding to the immobilized protein captured during the first incubation. After washing, a horseradish peroxidase labeled anti-rabbit IgG is added. This binds to the detection antibody to complete the four member sandwich. After a third incubation and washing to remove all the unbound enzyme, a substrate solution (TMB) is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of target protein present in the original specimen and the optical density can be read on a standard microplate reader.
Each manufactured lot of this ELISA kit is quality tested for criteria such as sensitivity, specificity, precision, and lot-to-lot consistency. See manual for more information on validation.
AMPK is a heterotrimeric complex comprising a catalytic alpha subunit and regulatory beta and gamma subunits. It protects cells from stresses that cause ATP depletion by switching off ATP-consuming biosynthetic pathways. AMPK is activated by high AMP and low ATP through a mechanism involving allosteric regulation, promotion of phosphorylation by an upstream protein kinase known as AMPK kinase, and inhibition of dephosphorylation. Activated AMPK can phosphorylate and regulate in vivo hydroxymethylglutaryl-CoA reductase and acetyl-CoA carboxylase, which are key regulatory enzymes of sterol synthesis and fatty acid synthesis, respectively. The human AMPK alpha1 and AMPK alpha2 genes encode 548 amino acid and 552 amino acid proteins, respectively. Human AMPK beta1 encodes a 271 amino acid protein and human AMPK beta2 encodes a 272 amino acid protein. The human AMPK gamma1 gene encodes a 331 amino acid protein. Human AMPK gamma2 and AMPK gamma3, which are 569 and 492 amino acid proteins, respectively, contain unique N-terminal domains and may participate directly in the binding of AMP within the AMPK complex.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.