| 货号 | 7179C |
| 描述 | CSTs PathScan® Total 4E-BP1 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of 4E-BP1. A 4E-BP1 Rabbit Antibody* has been coated onto the microwells. After incubation with cell lysates, 4E-BP1 (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a 4E-BP1 Mouse Detection Antibody* is added to detect the captured 4E-BP1 protein. Anti-mouse IgG, HRP-linked Antibody #7076* 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 total 4E-BP1. * Antibodies in kit are custom formulations specific to kit. |
| 反应种属 | Human |
| 应用 | ELISA |
| 目标/特异性 | CSTs PathScan® Total 4E-BP1 Sandwich ELISA Kit #7179 detects endogenous levels of 4E-BP1. As shown in Figure 1, using CSTs PathScan® Phospho-4E-BP1 (Thr37/46) Sandwich ELISA Kit #7216, a significant induction of 4E-BP1 phosphorylation at Thr37/46 is detected in serum and amino acid starved HEK-293T cells treated with insulin for 30 minutes after replenishing the amino acids. The level of total 4E-BP1 (phospho and nonphospho) remains unchanged as shown by Western analysis and by PathScan® Total 4E-BP1 Sandwich ELISA Kit #7179. 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 | Translation repressor protein 4E-BP1 (also known as PHAS-1) inhibits cap-dependent translation by binding to the translation initiation factor eIF4E. Hyperphosphorylation of 4E-BP1 disrupts this interaction and results in activation of cap-dependent translation (1). Both the PI3 kinase/Akt pathway and FRAP/mTOR kinase regulate 4E-BP1 activity (2,3). Multiple 4E-BP1 residues are phosphorylated in vivo (4). While phosphorylation by FRAP/mTOR at Thr37 and Thr46 does not prevent the binding of 4E-BP1 to eIF4E, it is thought to prime 4E-BP1 for subsequent phosphorylation at Ser65 and Thr70 (5). |
| 存放说明 | 4C |
| 参考文献 | Pause, A. et al. (1994) Nature 371, 762-7. Brunn, G.J. et al. (1997) Science 277, 99-101. Gingras, A.C. et al. (1998) Genes Dev 12, 502-13. Fadden, P. et al. (1997) J Biol Chem 272, 10240-7. Gingras, A.C. et al. (1999) Genes Dev 13, 1422-37. |
Figure 1: Treatment of HEK-293T cells with amino acids and insulin stimulates phosphorylation of 4E-BP1 at Thr37/46, detected by PathScan® Phospho-4E-BP1 (Thr37/46) Sandwich ELISA Kit #7216, but does not affect the level of total 4E-BP1 protein detected by PathScan® Total 4E-BP1 Sandwich ELISA Kit #7179. HEK-293T cells (70-80% confluent) were starved overnight and deprived of amino acids for 1 hour. The amino acids were replenished for 1 hour. Cells were either untreated or stimulated with 100 nM insulin for 30 minutes at 37ºC. λ phosphatase treatment of control cell lysates (4000 U/mL for 60 minutes at 37ºC) abolishes the basal phosphorylation of 4E-BP1 as shown by both sandwich ELISA and Western analysis. The absorbance readings at 450 nm are shown in the top figure while the corresponding Western blots, using 4E-BP1 Antibody #9452 (left panel) or Phospho-4E-BP1 (Thr37/46) Antibody #9459 (right panel), are shown in the bottom figure. 图1：使用氨基酸和胰岛素处理HEK-293T细胞系促使4E-BP1蛋白在Thr37/46位点磷酸化，随后使用PathScan® Phospho-4E-BP1 (Thr37/46) Sandwich ELISA Kit #7216检测，但是不影响通过PathScan® Total 4E-BP1 Sandwich ELISA Kit #7179分析4E-BP1总蛋白水平。HEK-293T细胞(70-80% confluent)饥饿培养过夜，接着氨基酸缺乏培养1小时。之后氨基酸重新加入培养1小时。随后细胞不处理或用100 nM胰岛素在37ºC处理30分钟。通过sandwich ELISA和Western blot分析显示对照组细胞裂解液(4000 U/mL在37ºC处理60分钟)的λ 磷酸酶处理终止了4E-BP1蛋白基本的磷酸化。450 nm的吸光度值显示在最上图中，然而通过使用4E-BP1 Antibody #9452 (左图)和Phospho-4E-BP1 (Thr37/46) Antibody #9459 (右图)，得到的western blot结果显示在下图中。 | |
Figure 2: The relationship between the protein concentration of the lysate from amino acid (AA)/untreated and AA/insulin-treated HEK-293T cells and the absorbance at 450 nm is shown. 图2：氨基酸(AA)/未处理和AA/胰岛素处理的HEK-293T细胞裂解物的蛋白浓度与在450 nm吸光度值之间的关系如图所示。 | |
Figure 3. Kit specificity as demonstrated by Western analysis of the ELISA microwell captured protein. Lysates were prepared from HEK-293T cells and incubated in microwells coated with the 4E-BP1 capture antibody. Wells were washed, and the captured protein was solubilized in SDS gel loading buffer. Western analysis of HEK-293T cell starting lysates (lanes 1 & 2) and the captured protein (lanes 3 & 4) was performed using 4E-BP1 Antibody #9452. The major band detected in the captured material (lanes 3 & 4) corresponds to 4E-BP1. 图3.Western blot分析验证ELISA微孔板捕获抗体的的特异性。用HEK-293T细胞制备裂解物，并且用4E-BP1捕获抗体的微孔中孵育。清洗微孔后，被捕获的蛋白溶解在SDS胶上样缓冲液中。使用4E-BP1 Antibody #9452进行Western blot分析HEK-293T细胞原始裂解物（第1,2泳道）和捕获到的蛋白（第3，4道）。被捕获物（第3，4道）上的主要条带与4E-BP1蛋白条带大小符合。 |
