Cell Signaling Technology

Product Pathways - Metabolism

PathScan® Phospho-AMPKα (Thr172) Chemiluminescent Sandwich ELISA Kit #7792

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Kit Includes Volume Solution Color
AMPKα Rabbit mAb Coated Microwells 96 tests
Phospho-AMPKα (T172) Mouse Detection Antibody 5.5 ml Green
Anti-mouse IgG, HRP-linked Antibody 5.5 ml Red
Luminol/Enhancer Solution 3 ml Colorless
Stable Peroxide Buffer 3 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
PathScan® Sandwich ELISA Lysis Buffer (1X) #7018 30 ml Colorless

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M

Reactivity Key:  H=Human  M=Mouse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

The PathScan® Phospho-AMPKα (Thr172) Chemiluminescent Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-AMPKα (Thr172) protein with a chemiluminescent readout. Chemiluminescent ELISAs often have a wider dynamic range and higher sensitivity than conventional chromogenic detection. This chemiluminescent ELISA, which is offered in low volume microplates, shows increased signal and sensitivity while using smaller samples. An AMPKα Rabbit mAb 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 the captured phospho-AMPKα (Thr172) protein. HRP-linked, anti-mouse antibody is then used to recognize the bound detection antibody. Chemiluminescent reagent is added for signal development. The magnitude of light emission, measured in relative light units (RLU), is proportional to the quantity of phospho-AMPKα (Thr172) protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

PathScan® Phospho-AMPKα (Thr172) Chemiluminescent Sandwich ELISA Kit detects endogenous levels of phospho-AMPKα (Thr172), as shown in figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA

ELISA

Figure 1: Relationship between protein concentration of lysates from phosphatase-treated and H2O2-treated C2C12 cells and immediate light generation with chemiluminescent substrate is shown. C2C12 cells (80-90% confluent) were treated with H2O2 (10 mM, 10 min, 37ºC). Graph inset corresponding to the shaded area shows high sensitivity and a linear response at the low protein concentration range.

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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Companion Products

Rabbit monoclonal antibody is produced under license (granting certain rights including those under U. S. Patents No. 5,675,063 and 7,429,487) from Epitomics, Inc.          

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

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