Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total 4E-BP1 Sandwich ELISA Kit #7179

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Kit Includes Volume Solution Color
4E-BP1 Ab Coated Microwells 96 tests
4E-BP1 Detection Ab 11 ml Green
Anti-mouse IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
Cell Lysis Buffer (10X) #9803 15 ml Yellowish
ELISA Sample Diluent 25 ml Blue

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

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.

ELISA - Western correlation

ELISA - Western correlation

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.

Sensitivity

Sensitivity

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.

Specificity

Specificity

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.


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, 762-767.
  2. Brunn, G.J. et al. (1997) Science 277, 99-101.
  3. Gingras, A.C. et al. (1998) Genes Dev. 12, 502-513.
  4. Fadden, P. et al. (1997) J. Biol. Chem. 272, 10240-10247.
  5. Gingras, A.C. et al. (1999) Genes Dev. 13, 1422-1437.

Application References

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Protocols

Companion Products

Rabbit Monoclonals are produced under license (granting certain rights, including those under U.S. Patent No. 5,675,063) from Epitomics, Inc.

For Research Use Only. Not For Use In Diagnostic Procedures.

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