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PathScan^® Phospho-AMPKα (Thr172) Sandwich ELISA Kit

PathScan^® Phospho-AMPKα (Thr172) Sandwich ELISA Kit #7959

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ELISA

Supporting Data

REACTIVITY

H M

Application Key:

ELISA-ELISA

Species Cross-Reactivity Key:

H-Human
M-Mouse

Product Information

Product Description

The PathScan^® Phospho-AMPKα (Thr172) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of AMPKα when phosphorylated at Thr172. An AMPKα rabbit antibody has been coated onto the microwells. After incubation with cell lysates, AMPKα (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a phospho-AMPKα (Thr172) mouse detection antibody is added to detect phosphorylation of Thr172 on the captured AMPKα protein. Anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of AMPKα phosphorylated at Thr172.

*Antibodies in this kit are custom formulations specific to kit.

Protocol

Specificity / Sensitivity

CST's PathScan^® Phospho-AMPKα (Thr172) Sandwich ELISA Kit #7959 detects AMPKα when phosphorylated at Thr172 as shown in Figure 1. Kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Species Reactivity:

Human, Mouse

Background

AMP-activated protein kinase (AMPK) is highly conserved from yeast to plants and animals and plays a key role in the regulation of energy homeostasis (1). AMPK is a heterotrimeric complex composed of a catalytic α subunit and regulatory β and γ subunits, each of which is encoded by two or three distinct genes (α1, 2; β1, 2; γ1, 2, 3) (2). The kinase is activated by an elevated AMP/ATP ratio due to cellular and environmental stress, such as heat shock, hypoxia, and ischemia (1). The tumor suppressor LKB1, in association with accessory proteins STRAD and MO25, phosphorylates AMPKα at Thr172 in the activation loop, and this phosphorylation is required for AMPK activation (3-5). AMPKα is also phosphorylated at Thr258 and Ser485 (for α1; Ser491 for α2). The upstream kinase and the biological significance of these phosphorylation events have yet to be elucidated (6). The β1 subunit is post-translationally modified by myristoylation and multi-site phosphorylation including Ser24/25, Ser96, Ser101, Ser108, and Ser182 (6,7). Phosphorylation at Ser108 of the β1 subunit seems to be required for AMPK activation, while phosphorylation at Ser24/25 and Ser182 affects AMPK localization (7). Several mutations in AMPKγ subunits have been identified, most of which are located in the putative AMP/ATP binding sites (CBS or Bateman domains). Mutations at these sites lead to reduction of AMPK activity and cause glycogen accumulation in heart or skeletal muscle (1,2). Accumulating evidence indicates that AMPK not only regulates the metabolism of fatty acids and glycogen, but also modulates protein synthesis and cell growth through EF2 and TSC2/mTOR pathways, as well as blood flow via eNOS/nNOS (1).

Database Links

UniProt ID: Q13131 , P54646

Entrez-Gene Id: 5562 , 5563

Limited Uses

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For Research Use Only. Not For Use In Diagnostic Procedures.

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PathScan® Phospho-AMPKα (Thr172) Sandwich ELISA Kit #7959