| 货号 | 7252C |
| 描述 | CSTs PathScan® Phospho-Akt (Thr308) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects phospho-Akt (Thr308) protein. An Akt Antibody has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-Akt proteins are captured by the coated antibody. Following extensive washing, Phospho-Akt (Thr308) Mouse mAb is added to detect the captured phospho-Akt 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 optical density for this developed color is proportional to the quantity of phospho-Akt (Thr308) protein. |
| 反应种属 | Human/Mouse |
| 应用 | ELISA |
| 目标/特异性 | CSTs PathScan® Phospho-Akt (Thr308) Sandwich ELISA Kit #7252 detects endogenous levels of phospho-Akt (Thr308) protein. As shown in Figure 1, using Phospho-Akt (Thr308) ELISA Kit #7252, a significant induction of Phospho-Akt (Thr308) is detected in NIH/3T3 cells treated with PDGF. However, levels of total Akt (phospho and nonphospho) detected by PathScan® Total Akt Sandwich ELISA Kit #7170, remain unchanged (Figure 1). Phospho-Akt (Thr308) in Jurkat cells is also detected by this ELISA kit #7252. 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, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (5,6). Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad (7), forkhead transcription factors (8), c-Raf (9), and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). LY294002 is a specific PI3 kinase inhibitor (11). Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (12,13). Akt may also play a role in insulin stimulation of glucose transport (12). In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip (15) and p21 Waf1/CIP1 (16). Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (17). More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex (18,19). |
| 存放说明 | 4C |
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Figure 1: Treatment of NIH/3T3 cells with PDGF stimulates phosphorylation of Akt at Thr308, detected by PathScan® Phospho-Akt (Thr308) Sandwich ELISA Kit #7252, but does not affect the level of total Akt detected by PathScan® Total Akt1 Sandwich ELISA Kit #7170. OD 450 nm readings are shown in the top figure, while the corresponding Western blots using Phospho-Akt (Thr308) Antibody #9275 (right panel) or Akt Antibody #9272 (left panel), are shown in the bottom figure.Figure 1，采用PDGF #9909 处理NIH/3T3细胞，使得Akt蛋白在苏氨酸308位点发生磷酸化，检测用试剂盒为PathScan ® Phospho-Akt (Thr308) Sandwich ELISA Kit #7252, 但没有影响总Akt1 ，采用by PathScan ® Total Akt1 Sandwich ELISA Kit #7170检测, ，在450nm处读取吸光度值（上图），相对应western blot采用抗体为Phospho-Akt (Thr308) Antibody #9275 (右泳道) 或 Akt Antibody #9272 (左泳道），见下图 | |
Figure 2: The relationship between protein concentration of lysates from untreated and PDGF-treated NIH/3T3 cells and kit assay optical density readings is shown. After starvation, NIH/3T3 cells (85% confluence) were treated with PDGF (50 ng/ml) for 10 min at 37°C, and then lysed.Firure2采用PDGF处理NIH/3T3细胞和未处理组细胞提取物中蛋白浓度与450nm处吸光度值的关系图。经过饥饿后， NIH/3T3 细胞（85%）采用PDGF (50 ng/ml ）37度处理10-15分钟，然后裂解。 |
