PathScan? Phospho-AMPKα (Thr172) Chemiluminescent Sandwich ELISA Kit

• 产品编号：CST-7792C      品牌：CST       原厂货号：7792C
• 产品分类：ELISA及其它免疫分析 > ELISA > ELISA检测试剂盒
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描述：

PathScan® Phospho-AMPKα (Thr172) Chemiluminescent Sandwich ELISA Kit 能够检测内源性磷酸化AMPKα (Thr172)蛋白水平，如图1所示。本试剂盒可检测经内部测试确定的指定物种的蛋白质，同时也可以检测其他物种的同源蛋白。PathScan® Phospho-AMPKα (Thr172) Chemiluminescent Sandwich ELISA Kit采用了固相夹心酶联免疫吸附方法（ELISA）通过化学发光读数检测内源性磷酸化-AMPKα (Thr172)蛋白水平。与传统的显色检测法相比，化学发光ELISAs往往有着更宽泛的动态范围和更高的灵敏度。这种化学发光ELISA采用低体积的微孔板，在加强信号和灵敏度的同时需要的样本量更少。微孔中包被了AMPKα Rabbit mAb兔单抗。细胞裂解液孵育后，（磷酸化及非磷酸化）AMPKα被包被的抗体捕获。充分洗涤后，添加Phospho-AMPKα (Thr172) Mouse Detection Antibody检测捕获的磷酸化-AMPKα (Thr172)蛋白。再用HRP-标记的抗小鼠的抗体检测结合的抗体。添加化学发光试剂增强信号。测得的相对光单位（RLU）与磷酸化-AMPKα (Thr172)蛋白的量成正比。试剂盒中的抗体为该试剂盒所特有。AMP依赖的蛋白激酶（AMPK）从酵母到植物和动物都高度保守，在调节能量平衡方面起关键作用（1）。 AMPK蛋白以一种异源三聚体复合物的形式存在，由一个α-催化亚基、一个β-调节亚基和一个γ-调节亚基组成，每个亚基被两到三个不同基因编码（α1，β1，γ1，2，3）（2）。该激酶会被因细胞和环境压力引起的AMP/ ATP比值升高而激活，如热休克，缺氧和缺血（1）。肿瘤抑制因子LKB1与辅助蛋白STRAD和MO25一同将位于AMPKα活化环的Thr172磷酸化，该位点的磷酸化是AMPK激酶活性所必需的（3-5）。 AMPKα的Thr258和Ser485（α1是Ser485，α2是Ser491）也被磷酸化。上游激酶以及这些磷酸化事件的生物学意义尚未被阐明（6）。β1亚基是被翻译后修饰的，如肉豆蔻酰化和多位点磷酸化包括Ser24/25，Ser96，Ser101，Ser108和Ser182（6,7）。β1亚基Ser108磷酸化似乎是需要AMPK激酶活性的，而Ser24/25和Ser182磷酸化会影响AMPK定位（7）。已确定了几个AMPKγ亚基的突变，其中大部分是位于假定AMP/ ATP结合位点（CBS或贝特曼域）。在这些位点的突变导致AMPK活性降低，引起心脏或骨骼肌内糖原积累（1,2）。越来越多的证据表明，AMPK不仅调节脂肪酸和糖原代谢，也通过EF2和TSC2/mTOR途径调节蛋白质合成和细胞生长，另外还通过eNOS / nNOS调节血流量（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.

原厂资料：

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.

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.

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.

注意事项：

Low volume microplate * 12 8-well modules -each module is designed to break apart for 8 tests.
**Kit should be stored at 4°C with the exception of 10X Cell Lysis Buffer, which is stored at –20°C (packaged separately).