Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-AMPKα (Thr172) Sandwich ELISA Antibody Pair #7955

Kit Includes Volume Cap Color
AMPK-alpha Capture Ab (100X) 0.4 milliliters Pink
Phospho-AMPK-alpha (Thr172) Detection Ab (100X)(biotinylated) 0.4 milliliters Blue
HRP-linked Streptavidin(1000X) 0.04 milliliters Natural

Capture and detection antibodies are stored at 4°C. HRP-linked secondary reagent is stored at -20°C.

Species Cross-Reactivity

H M

Reactivity Key:  H=Human  M=Mouse

Description

CST's PathScan® Phospho-AMPKα (Thr172) Sandwich ELISA Antibody Pair is being offered as an economical alternative to our PathScan® Phospho-AMPKα-(Thr172) Sandwich ELISA Kit #7959. Capture and Detection antibodies (100X stocks) and HRP-conjugated streptavidin (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The AMPKα Capture Antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysates are added followed by a biotinylated Phospho-AMPKα (Thr172) Detection Antibody and HRP conjugated streptavidin. HRP substrate, TMB, is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of Phospho-AMPKα (Thr172) protein.*Antibodies in this kit are custom formulations specific to the kit.

ELISA

ELISA

Figure 1. The relationship between the protein concentration of lysates from untreated and H2O2-treated C2C12 cells and the absorbance at 450 nm using the PathScan® Phospho-AMPKα (Thr172) Sandwich ELISA Antibody Pair #7955 is shown.

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 the activation of AMPK enzyme, 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).

  1. Hardie, D.G. (2004) J Cell Sci 117, 5479-87.
  2. Carling, D. (2004) Trends Biochem Sci 29, 18-24.
  3. Hawley, S.A. et al. (1996) J Biol Chem 271, 27879-87.
  4. Lizcano, J.M. et al. (2004) EMBO J 23, 833-43.
  5. Shaw, R.J. et al. (2004) Proc Natl Acad Sci USA 101, 3329-35.
  6. Woods, A. et al. (2003) J Biol Chem 278, 28434-42.
  7. Warden, S.M. et al. (2001) Biochem J 354, 275-83.

Application References

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This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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