PathScan? Total 4E-BP1 Sandwich ELISA Kit

• 产品编号：CST-7179C      品牌：CST       原厂货号：7179C
• 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：

CST's PathScan® Total 4E-BP1 Sandwich ELISA Kit #7179仅检测的内源性phospho-4E-BP1蛋白。如图1所示，使用CST's PathScan® Phospho-4E-BP1 (Thr37/46) Sandwich ELISA Kit #7216检测，HEK-293T在无血清和缺乏氨基酸培养，随后重新加入氨基酸并给予胰岛素处理30分钟，显示Thr37/46位点磷酸化的4E-BP1蛋白显著增加。通过Western blot和PathScan® Total 4E-BP1 Sandwich ELISA Kit #7179分析，4E-BP1(磷酸化和非磷酸化)总蛋白水平仍然没有改变。通过内部实验确定，该试剂盒检测指定的物种，但是可能也会检测其它物种的同源性蛋白。该CST's PathScan® Total 4E-BP1 Sandwich ELISA Kit是一个固相夹心酶联免疫吸附法，其能检测内源性4E-BP1蛋白。4E-BP1 Rabbit Antibody已经被包被在微孔板上。在与细胞裂解液一起孵育后，4E-BP1 (磷酸化和非磷酸化)蛋白被包被的抗体吸附。在充分清洗后，加入4E-BP1鼠源检测抗体以检测被吸附的4E-BP1蛋白。然后，Anti-mouse IgG, HRP-linked Antibody #7076被用于识别已结合的检测抗体。加入HRP底物TMB后显色。该颜色的吸光度值的高低与4E-BP1总蛋白多少成正比。试剂盒里的抗体是专门针对该试剂盒定制的。翻译抑制蛋白4E-BP1 (又称 PHAS-1)通过结合翻译起始因子eIF4E蛋白从而抑制帽子结构依赖的翻译。4E-BP1的过磷酸化干扰上述结合并且导致激活帽子结构依赖的翻译（1）。PI3 kinase/Akt通路和FRAP/mTOR激酶通路都调节4E-BP1蛋白的激活（2,3）。在体内4E-BP1多个位点残基被磷酸化（4）。通过FRAP/mTOR蛋白使4E-BP1在Thr37和Thr46位点磷酸化不能阻止它eIF4E结合，但这被认为是为随后在4E-BP1的Ser65和Thr70位点磷酸化进行准备（5）。

1. Pause, A. et al. (1994) Nature 371, 762-7.
2. Brunn, G.J. et al. (1997) Science 277, 99-101.
3. Gingras, A.C. et al. (1998) Genes Dev 12, 502-13.
4. Fadden, P. et al. (1997) J Biol Chem 272, 10240-7.
5. Gingras, A.C. et al. (1999) Genes Dev 13, 1422-37.

原厂资料：

Specificity / Sensitivity

CST's PathScan® Total 4E-BP1 Sandwich ELISA Kit #7179 detects endogenous levels of 4E-BP1. As shown in Figure 1, using CST's 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.

Description

CST's 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.

Background

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).

1. Pause, A. et al. (1994) Nature 371, 762-7.
2. Brunn, G.J. et al. (1997) Science 277, 99-101.
3. Gingras, A.C. et al. (1998) Genes Dev 12, 502-13.
4. Fadden, P. et al. (1997) J Biol Chem 272, 10240-7.
5. Gingras, A.C. et al. (1999) Genes Dev 13, 1422-37.

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).