PathScan? Cell Growth Multi-Target Sandwich ELISA Kit

• 产品编号：CST-7239S      品牌：CST       原厂货号：7239S
• 产品分类：ELISA及其它免疫分析 > ELISA > ELISA检测试剂盒
• 应用分类：

其它组分：

描述：

CST的PathScan ® Total Akt1 Sandwich ELISA Kit#7239可以检测内源性的六种蛋白：S6核糖体蛋白，磷酸化S6核糖体蛋白（Ser235/236），AKT1，磷酸化AKT（ser473），磷酸化AKT（Thr308）和磷酸化p44/42MAPK（Thr202/Tyr204）。PDGF刺激后，随着时间的增加可以观察到这些蛋白的激活。如Figrue 1中所示，用PDGF刺激血清饥饿的NIH/3T3细胞促进Akt1的Thr308和Ser473位的磷酸化，S6核糖体蛋白的Ser235/236位的磷酸化，p44/42MAPK在Thr202/Tyr204位的磷酸化。Western实验显示这些靶标蛋白的水平（磷酸化和非磷酸化）在80分钟内都保持不变。蛋白裂解液浓度的联系和在450nm的吸光值可以在每个PathScan® Sandwich ELISA Kits相关的数据表中找到。本试剂盒经内部测试后显示可检测指定物种的蛋白，但也可能检测出其它物种的同源蛋白。CST的PathScan® PathScan® Cell Growth Multi-Target Sandwich ELISA Kit 是一种固相三明治酶联免疫吸附方法（ELISA），其中集合了用来检测S6核糖体蛋白，磷酸化S6核糖体蛋白（Ser235/236），AKT1，磷酸化AKT（Ser473），磷酸化AKT（Thr308）和磷酸化p44/42MAPK（Thr202/Tyr204）。这些分子代表了控制生长和分化通路的关键信号蛋白。每个目标蛋白都提供16个检测。对于指定目标蛋白的特定检测配方可以在每个三明治Elisa试剂盒相关的数据表中查到。简单的来说，一个捕获抗体抗体**已经包被在孔中。在与细胞的裂解液共孵育后，目标蛋白被已包被的抗体捕获。经过彻底的洗涤，加入检测抗体**来检测所捕获的靶蛋白。HRP标记的二抗用来识别结合的检测抗体。ＨＲＰ底物，ＴＭＢ加入后产生颜色。所产生颜色的吸光度值与结合的目标蛋白的数量呈正相关。*见配套产品。**试剂盒中的抗体是根据所需成分特别定制的。Akt是一种原癌基因 ，在多种细胞活动如细胞的生长，存活及细胞周期中起着至关重要的调节作用。Akt也是胰岛素信号和糖代谢的主要调节因子（1-4）。Akt可以被PI3激酶信号激活，包括在Thr308位点的激活环被PDK1磷酸化，以及在羧基端Ser473位点被mTOR-rictor复合体(TORC1)磷酸化(5-7)。 P44和p42 MAP激酶(Erk1 和 Erk2)都具有蛋白激酶级联功能，在细胞生长和分化中起着关键的作用（8-13）。 MAP激酶可以被很多细胞间信号激活包括生长因子和神经营养因子，细胞因子，激素和神经递质。MAP激酶通过一个上游MAP激酶的激酶（MEK）来磷酸化其T*EY*序列上的苏氨酸和酪氨酸(人类MAP激酶的202 和 204 或 大鼠MAP 激酶的183 和 185 )从而被激活(14,15)。 为了有效且持续的促进细胞生长和分裂，生长因子和丝裂原必须上调翻译（16,17）。生长因子和丝裂原诱导p70 S6 激酶的活化，进而使S6核糖体糖蛋白发生磷酸化。 S6核糖体糖蛋白的磷酸化与翻译的增加相关联，尤其是在5’非翻译区域带有寡嘧啶的mRNA（17）。这种mRNA (5’TOP)编码参与细胞周期的进程的蛋白和翻译复合物的组成蛋白如核糖体蛋白和延伸因子的蛋白（17,18）。体内S6核糖体蛋白主要的磷酸化位点包括Ser235, Ser236, Ser240 和 Ser244，它们位于S6羧基末端的一个小的包含19个氨基酸的区域内（19,20）。

原厂资料：

Specificity / Sensitivity

CST's PathScan® Cell Growth Multi-Target Sandwich ELISA Kit #7239 detects endogenous levels of six proteins: S6 ribosomal protein, phospho-S6 ribosomal protein (Ser235/236), Akt1, phospho-Akt (Ser473), phosho-Akt (Thr308) and phospho-p44/42 MAPK (Thr202/Tyr204). Activation of these proteins can be observed over time in response to PDGF. As shown in Figure 1, stimulation of serum-starved NIH/3T3 cells with PDGF promotes phosphorylation of Akt1 at Thr308 and Ser473, S6 ribosomal protein at Ser235/236 and p44/42 MAPK at Thr202/Tyr204. The level of each target protein (phospho and nonphospho) remains unchanged throughout the 80 minute time course as demonstrated by Western analysis. 
 The relationship between the protein concentration of the lysate and the absorbance at 450 nm can be found in the datasheets associated with the individual PathScan® Sandwich ELISA Kits*. *See companion products. 
 This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Description

CST’s PathScan® Cell Growth Multi-Target Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that combines the reagents necessary to detect endogenous levels of S6 ribosomal protein, phospho-S6 ribosomal protein (Ser235/236), Akt1, phospho-Akt (Ser473), phospho-Akt (Thr308) and phospho-p44/42 MAPK (Thr202/Tyr204). These molecules represent key signaling proteins in pathways controlling growth and differentiation. Sixteen assays are provided for each target protein. Specific assay formulations for the indicated target proteins can be found in the datasheets associated with the individual sandwich ELISA kits*. Briefly, a capture antibody** has been coated onto the microwells. After incubation with cell lysates, the target protein is captured by the coated antibody. Following extensive washing, a detection antibody** is added to detect the captured target protein. An HRP-linked secondary antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of bound target protein. *See companion products. **Antibodies in kit are custom formulations specific to kit.

Background

Akt is a protooncogene with a critical regulatory role in diverse cellular processes including growth, survival and the cell cycle. Akt is also a major regulator of insulin signaling and glucose metabolism (1-4). Akt is activated by PI3 kinase signaling and activation loop phosphorylation at Thr308 by PDK1 and by phosphorylation within the carboxy terminus at Ser473 by the mTOR-rictor complex (TORC1) (5-7). 
 Both p44 and p42 MAP kinases (Erk1 and Erk2) function in a protein kinase cascade that plays a critical role in the regulation of cell growth and differentiation (8-13). MAP kinases are activated by a wide variety of extracellular signals including growth and neurotrophic factors, cytokines, hormones and neurotransmitters. Activation of MAP kinases occurs through phosphorylation of threonine and tyrosine (202 and 204 of human MAP kinase or 183 and 185 of rat MAP kinase) at the sequence T*EY* by a single upstream MAP kinase kinase (MEK) (14,15). 
 To effectively promote growth and cell division in a sustained manner, growth factors and mitogens must upregulate translation (16,17). Growth factors and mitogens induce the activation of p70 S6 kinase, which in turn phosphorylates the S6 ribosomal protein. Phosphorylation of S6 ribosomal protein correlates with an increase in translation, particularly of mRNAs with an oligopyrimidine tract in their 5' untranslated regions (17). This group of mRNAs (5'TOP) encodes proteins involved in cell cycle progression and proteins that are part of the translational machinery, such as ribosomal proteins and elongation factors (17,18). The main in vivo S6 ribosomal protein phosphorylation sites, including Ser235, Ser236, Ser240 and Ser244, are located within a small 19 amino acid region in the S6 carboxy terminus (19,20).

注意事项：

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).