Revision 7

#7179Store at +4C

1 个试剂盒

(96 assays)

Species Cross Reactivity

H

UniProt ID:

#Q13541

Entrez-Gene Id:

#1978

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Color Storage Temp
4E-BP1 Rabbit mAb Coated Microwells 32193 96 tests +4C
4E-BP-1 Mouse Detection mAb 9463 1 ea Green (Lyophilized) +4C
Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated) 13304 1 ea Red (Lyophilized) +4C
Detection Antibody Diluent 2 14621 11 ml Green +4C
HRP Diluent 13515 11 ml Red +4C
TMB Substrate 7004 11 ml +4C
STOP Solution 7002 11 ml +4C
Sealing Tape 54503 2 ea +4C
ELISA Wash Buffer (20X) 9801 25 ml +4C
ELISA Sample Diluent 11083 25 ml Blue +4C
Cell Lysis Buffer (10X) 9803 15 ml -20C

*The microwell plate is supplied as 12 8-well modules - Each module is designed to break apart for 8 tests.

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.

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.

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.

Background References

    Cross-Reactivity Key

    H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    PathScan is a registered trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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    Revision 7
    #7179

    PathScan® Total 4E-BP1 Sandwich ELISA Kit

    PathScan® Total 4E-BP1 Sandwich ELISA Kit: Image 1 Expand Image
    图 1:HEK-293T 细胞用氨基酸和胰岛素处理会刺激4E-BP1 在Thr37/46位点的磷酸化,可使用 PathScan® Phospho-4E-BP1 (Thr37/46) Sandwich ELISA Kit #7216 进行检测,但不影响 PathScan® Total 4E-BP1 Sandwich ELISA Kit #7179 检测的 4E-BP1 总蛋白的水平。HEK-293T 细胞(70-80% 融合度)饥饿过夜,并剥夺氨基酸 1 小时。补充氨基酸 1 小时。细胞未经处理或用 100 nM 胰岛素在 37ºC 下刺激 30 分钟。夹心 ELISA 和蛋白质印迹分析表明,对照的细胞裂解物用 λ 磷酸酶处理(4000 U/mL,60 分钟,在 37ºC 下)会减弱 4E-BP1 基础磷酸化。在 450 nm 处的吸光度读数如顶部图所示,使用 4E-BP1 Antibody #9452(左小图)或 Phospho-4E-BP1 (Thr37/46) Antibody #9459(右小图)检测到的相应蛋白质印迹如底部图所示。
    PathScan® Total 4E-BP1 Sandwich ELISA Kit: Image 2 Expand Image
    图 2:氨基酸 (AA)/未经处理的和已经 AA/胰岛素处理的 HEK-293T 细胞裂解物蛋白浓度与在 450 nm 处的吸光度之间的关系如图所示。
    PathScan® Total 4E-BP1 Sandwich ELISA Kit: Image 3 Expand Image
    图 3. 对 ELISA 微孔捕获的蛋白进行蛋白质印迹分析证实了试剂盒的特异性。裂解物用 HEK-293T 细胞制备,并在 4E-BP1 捕获抗体包被的微孔中孵育。洗涤各孔,捕获的蛋白在 SDS 凝胶上样缓冲液中进行溶解。使用 4E-BP1 Antibody #9452 对 HEK-293T 细胞的初步裂解物(泳道 1 和 2)和捕获蛋白(泳道 3 和 4)进行蛋白质印迹分析。在捕获物质(泳道 3 和 4)中检测到的主要条带对应于 4E-BP1。