| 货号 | 7239S |
| 描述 | 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. |
| 反应种属 | Human/Mouse |
| 应用 | ELISA |
| 目标/特异性 | CSTs 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. |
| 供应商 | CST |
| 背景 | 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 (5TOP) 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).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）。 |
| 存放说明 | 4C |
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Figure 1. Treatment of NIH/3T3 cells with PDGF induces phosphorylation of Akt1 at Thr308 and Ser473, S6 Ribosomal Protein at Ser235/236 and p44/42 MAPK at Thr202/Tyr204 as detected by the PathScan® Cell Growth Multi-Target Sandwich ELISA Kit #7239. While dynamic phosphorylation is observed throughout the time course, the level of total p44/42 MAPK, Akt1 and S6 ribosomal protein remains unchanged as demonstrated by sandwich ELISA and Western analysis. NIH/3T3 cells (80-90% confluent) were starved overnight and stimulated with PDGF (100 ng/mL) for 5, 10, 20, 40 and 80 minutes at 37ºC. Lysates were assayed at a protein concentration of 0.45 mg/mL. The absorbance readings at 450 nm are shown as a 3-dimensional representation in the left figure, while the corresponding Western blots are shown in the right figure. The antibodies used for the Western analyses include S6 Ribosomal Protein Rabbit mAb #2217, Phospho-S6 Ribosomal Protein (Ser235/236) Antibody #2211, Akt Antibody #9272, Phospho-Akt (Ser473) (193H12) Rabbit mAb #4058, Phospho-Akt (Thr308) Antibody #9275, Phospho-p44/42 MAPK (Thr202/Tyr204) (E10) Mouse mAb #9106, p44/42 MAP Kinase Antibody #9102. Figure 1 NIH/3T3细胞在PDGF处理后，诱导的Akt1 在 Thr308 和 Ser473, S6 Ribosomal Protein 在Ser235/236 和p44/42 MAPK 在Thr202/Tyr204的磷酸化，检测用PathScan ® Cell Growth Multi-Target Sandwich ELISA Kit #7239 。整个时间里动态磷酸化， p44/42 MAPK, Akt1和S6核糖体蛋白的总磷酸化经ELISA和western blot分析后保持不变。NIH/3T3 细胞（80-90%）饥饿过夜后，采用 PDGF (100 ng/mL）在37°C处理5, 10, 20, 40 和 80 分钟 。蛋白浓度为 0.45 mg/mL提取细胞裂解液。450nm的吸光度值在左侧图中用三维图标表示。相应的western blot结果在右侧图中。采用的抗体包括e S6 Ribosomal Protein Rabbit mAb 兔单抗#2217, Phospho-S6 Ribosomal Protein (Ser235/236) Antibody #2211, Akt Antibody #9272, Phospho-Akt (Ser473) (193H12) Rabbit mAb兔单抗 #4058, Phospho-Akt (Thr308) Antibody #9275, Phospho-p44/42 MAPK (Thr202/Tyr204) (E10) Mouse mAb鼠单抗 #9106, p44/42 MAP Kinase Antibody #9102。 | |
Figure 2. Schematic representation of a 96-well plate depicting the color-code of the reagents used to detect endogenous levels of Akt1 (red; 1 & 2), phospho-Akt1 (Ser473) (tan; 3 & 4), phospho-Akt1 (Thr308) (blue; 5 & 6), phospho-p44/42 MAPK (Thr202/Tyr204) (light pink; 7 & 8), S6 ribosomal protein (purple; 9 & 10) and phospho-S6 ribosomal protein (Ser235/236) (green; 11 & 12).Figure2，图解显示96孔板中不同颜色检测Akt1 (red; 1 & 2), phospho-Akt1 (Ser473) (tan; 3 &4), phospho-Akt1 (Thr308) (blue; 5 & 6), phospho-p44/42 MAPK (Thr202/Tyr204) (light pink; 7 & 8), S6 ribosomal protein (purple; 9 & 10) 和 phospho-S6 ribosomal protein (Ser235/236) (green; 11 & 12)的内源性水平。 |
