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.Learn more about how we get our images
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
1X Cell Lysis Buffer: PathScan® Sandwich ELISA Lysis Buffer (#7018) 1X: This buffer is ready to use as is. Buffer can be stored at 4°C for short-term use (1–2 weeks).
Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.
STOP Solution: (#7002)
NOTE: Reagents should be made fresh daily.
posted January 2008
revised January 2016
|Product Includes||Volume||Cap Color|
|AMPKα Rabbit Capture Rabbit mAb (100X)||400 µl||Pink|
|Phospho-AMPKα (Thr172) Detection Mouse mAb (100X)||400 µl||Blue|
|Anti-mouse IgG, HRP-linked Antibody (1000X)||40 µl||Yellow|
CST's PathScan® Phospho-AMPKα (Thr172) Sandwich ELISA Antibody Pair is offered as an economical alternative to our PathScan® Phospho-AMPKα-(Thr172) Sandwich ELISA Kit #7959. Capture and Detection antibodies (100X stocks) and Anti-Mouse IgG, HRP-linked Antibody (1000X stock) are supplied. Sufficient reagents are provided for 4 x 96 well ELISAs. The AMPKα Rabbit Capture Antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysates are added followed by a Phospho-AMPKα (Thr172) Mouse Detection Antibody and Anti-Mouse IgG, HRP-linked Antibody. 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 kit are custom formulations specific to kit.
Capture and detection antibodies are stored at 4°C. HRP-linked secondary reagent is stored at -20°C.
For Antibody Pair specificity and sensitivity, please refer to the corresponding PathScan® Sandwich ELISA Kit. Note: This antibody pair 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).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. PathScan is a trademark of Cell Signaling Technology, Inc. U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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|7955S||1 Kit (Reagents for 4 x 96 well plates)||N/A|
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