The phospho 4EBP1 (Thr37/46) kit measures cellular phospho-4EBP1, phosphorylated at Thr37/46, as an mTOR readout in the translational control pathway.
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  • Highly specific Highly specific
  • Ease-of-use Ease-of-use
The phospho 4EBP1 (Thr37/46) kit measures cellular phospho-4EBP1, phosphorylated at Thr37/46, as an mTOR readout in the translational control pathway.
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Overview

The phospho-4EBP1 assay kit is designed for the quantification of endogenous 4EBP1 protein, phosphorylated on Thr37/46. Based on the TR-FRET technology, the HTRF phospho-4EBP1 sandwich immunoassay provides an ideal readout solution in any cell type, for mTOR signaling and the translational control pathway. The optimized no-wash protocol facilitates mechanistic signaling pathway studies, analysis of compound mechanism of action, and target research, by delivering enhanced convenience and more relevant, reliable results.

Benefits

  • DETECTS ENDOGENOUS LEVELS
  • VALIDATED ON MULTIPLE CELL LINES

Phospho-4EBP1 (Thr37/46) Assay principle

The Phospho-4EBP1 (Thr37/46) assay measures 4EBP1 when phosphorylated at Thr37/46. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-4EBP1 (Thr37/46) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.
Phospho-4EBP1 (Thr37/46) Assay principle

Phospho-4EBP1 (Thr37/46) 2-plate Assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Phospho-4EBP1 (Thr37/46) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-4EBP1 (Thr37/46) 2-plate Assay protocol

Phospho-4EBP1 (Thr37/46) 1-plate assay protocol

Detection of Phosphorylated 4EBP1 (Thr37/46) with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Phospho-4EBP1 (Thr37/46) 1-plate Assay protocol

Phospho-4EBP1 HTRF assay performance compared to WB

Human HEK293 cells were grown in a T175 flask at 37°C until 80% confluency. After removal of cell culture medium, 3 mL of supplemented lysis buffer were added and incubated for 30 minutess. Soluble supernatants were collected after a 10 minute centrifugation. Equal amounts of lysates were used for a side by side comparison of Western Blot and HTRF. Using HTRF phospho-4EBP1 (THR37/46), just 400 cells are sufficient for minimal signal detection while 11,300 cells are needed for a Western Blot signal. The HTRF assay is 32-fold more sensitive than the Western Blot
Phospho-4EBP1 HTRF assay performance compared to Western Blot

Phospho-4EBP1 response upon mTORC1 activation

Jurkat, A431 and SW480 cells were seeded at 25,000 cells per well in a 96-well plate with DMEM+10%FCS for 24 hours. The cells were treated for 3 hours with known pan-mTOR inhibitors, either with increasing concentrations of PP242 or BEZ235. The two-plate adherent protocol was applied to A431 and SW480 cells, whereas the two-plate suspension protocol was followed for Jurkat cells.
Pharmacological response on phospho-4EBP1 inhibition of mTORC1
Pharmacological response on phospho-4EBP1 inhibition of mTORC1

Species compatibility: PP242 + Rapamycin in murine NIH3T3 cells

Murine NIH3T3 cells were incubated with various concentrations of tPP242 and Rapamycin, followed by stimulation with Insulin for 30 minutess at 37°C. After lysis, inhibition of 4EBP1 phosphorylation was measured using the HTRF phospho-4EBP1 (THR37/46) assay according to the two-plate protocol. PP242 is a potent pan-mTOR inhibitor of both mTORC1 (including Rapamycin-resistant 4EBP phosphorylation) and mTORC2, whereas Rapamycin treatment is known to have only partial or no effect on 4EBP1 phosphorylation.
Species compatibility: PP242 + Rapamycin in murine NIH3T3 cells

Simplified pathway of 4EPB1 cell signaling

Translation is initiated when a ribosome is recruited to the 5’ end of an mRNA. The eIF4E-binding proteins, such as 4EBP1, block translation by binding to eIF4E and preventing recruitment of the translation machinery. Hypophosphorylated 4EBP1 downregulates translation, whereas 4EBP1 hyperphosphorylation by mTOR abrogates eIF4E-binding activity and therefore promotes translation. The assessment of cellular phospho-4EBP1 is an important tool to generally measure mTOR potency and to determine the activity of mTOR signaling.
4EPB1 cell signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

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