Revision 4

#66753

Store at +4C

FastScan™ Phospho-4E-BP1 (Ser65) ELISA Kit

1 Kit

(96 assays)

Species Cross Reactivity:
H

UniProt ID:
#Q13541

Entrez-Gene Id:
#1978

CST Logo
Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

Web:

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Includes Product # Quantity Color
FastScan™ ELISA Microwell Strip Plate, 96 Well5325796 tests
4E-BP1 Rabbit Capture mAb353991 eaGreen (Lyophilized)
Phospho-4E-BP1 (Ser65) Rabbit HRP-linked mAb528151 eaRed (Lyophilized)
FastScan™ ELISA Capture Antibody Diluent160763 mlGreen
FastScan™ ELISA HRP Antibody Diluent281203 ml
TMB Substrate700411 ml
STOP Solution700211 ml
Sealing Tape545031 ea
ELISA Wash Buffer (20X)980125 ml
FastScan™ ELISA Cell Extraction Buffer (5X)6990510 ml
FastScan™ ELISA Cell Extraction Enhancer Solution (50X)252431 ml
FastScan™ ELISA Kit #66753 Positive Control Type 1930721 ea

Kit contents scale proportionally with size, except sealing tape.
Example: The V1 kit contains 5X the listed quantities above, but will exclude the sealing tape.

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

Description

The FastScan™ Phospho-4E-BP1 (Ser65) ELISA Kit is a sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of 4E-BP1 when phosphorylated at Ser65. To perform the assay, sample is incubated with a capture antibody conjugated with a proprietary tag and a second detection antibody linked to HRP, forming a sandwich with phospho-4E-BP1 (Ser65) in solution. This entire complex is immobilized to the plate via an anti-tag antibody. The wells are then washed to remove unbound material. TMB is then added. The magnitude of observed signal is proportional to the quantity of phospho-4E-BP1 (Ser65).

*Antibodies in this kit are custom formulations specific to kit.

Specificity/Sensitivity

The FastScan™ Phospho-4E-BP1 (Ser65) ELISA Kit detects endogenous levels of 4E-BP1 when phosphorylated at Ser65, as shown in Figure 1. 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).

Background References

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

FastScan™ ELISA is a registered trademark of Cell Signaling Technology, Inc.

U.S. Patents 9,086,407, 9,261,500, and 9,476,874, foreign equivalents, and child patents deriving therefrom.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] • Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 4

#66753

FastScan™ Phospho-4E-BP1 (Ser65) ELISA Kit

Cell Signaling Technology Logo
Figure 1.Treatment of MCF7 cells with amino acid (AA) replenishment increases the phosphorylation of 4E-BP1 at Ser65 but does not affect the level of total 4E-BP1. The relationship between lysate protein concentration from untreated and AA-treated MCF7 cells and the absorbance at 450 nm using the FastScan™ Phospho-4E-BP1 (Ser65) ELISA Kit #66753 is shown in the upper figure. The corresponding western blots using phospho-4E-BP1 (Ser65) antibody (left panel) and 4E-BP1 antibody (right panel) are shown in the lower figure. After overnight serum starvation, MCF7 cells were amino acid starved for 1 hour. Cells were either untreated or treated with amino acid replenishment for 1 hour at 37°C, and then lysed.
ELISA Image 1: FastScan<sup>™</sup> Phospho-4E-BP1 (Ser65) ELISA Kit
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] • Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.