Figure 2. The relationship between the protein concentration of lysates from untreated, phosphatase-treated and H2O2-treated C2C12 cells and the absorbance at 450 nm using the PathScan® Phospho-AMPKα (Thr172) Sandwich ELISA Kit is shown.
Figure 1. Treatment of C2C12 cells with H2O2 stimulates phosphorylation of AMPKα at Thr172, detected by the PathScan® Phospho-AMPKα (Thr172) Sandwich ELISA Kit #7959. C2C12 cells (80-90% confluent) were treated with 10 mM H2O2 for 10 minutes at 37ºC. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using AMPKα (23A3) Rabbit mAb #2603 (left panel) or Phospho-AMPKα (Thr172) (D79.5E) XP® Rabbit mAb #4188 (right panel) are shown in the bottom figure.
|Product Includes||Volume||Solution Color|
|AMPKα Rabbit mAb Coated Microwells||96 tests|
|Phospho-AMPKα (Thr172) Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 ea|
|PathScan® Sandwich ELISA Lysis Buffer (1X) 7018||30 ml|
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 kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
revised January 2016
Protocol Id: 205
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.
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).
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PathScan is a trademark of Cell Signaling Technology, Inc.