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 enclosed in parentheses are predicted to react based on 100% sequence homology.
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
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.
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.
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.
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).
- Pause, A. et al. (1994) Nature 371, 762-767.
- Brunn, G.J. et al. (1997) Science 277, 99-101.
- Gingras, A.C. et al. (1998) Genes Dev. 12, 502-513.
- Fadden, P. et al. (1997) J. Biol. Chem. 272, 10240-10247.
- Gingras, A.C. et al. (1999) Genes Dev. 13, 1422-1437.
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- 7216 PathScan® Phospho-4E-BP1 (Thr37/Thr46) Sandwich ELISA Kit
- 9644 4E-BP1 (53H11) Rabbit mAb
- 9452 4E-BP1 Antibody
- 2855 Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb
- 9459 Phospho-4E-BP1 (Thr37/46) Antibody
- 7076 Anti-mouse IgG, HRP-linked Antibody
- 9803 Cell Lysis Buffer (10X)
- 7002 STOP Solution
- 7004 TMB Substrate
- 9808 Phosphate Buffered Saline (PBS-20X)
- 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)
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.