The phospho-EIF4E (Ser209) kit enables the cell-based quantification of EIF4E protein phophorylated at Ser209 as a readout of mTor pathway activation.
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  • Highly specific Highly specific
  • Ease-of-use Ease-of-use
  • High sensitivity High sensitivity
The phospho-EIF4E (Ser209) kit enables the cell-based quantification of EIF4E protein phophorylated at Ser209 as a readout of mTor pathway activation.
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Overview

The Phospho-EIF4E (Ser209) cellular assay kit measures endogenous EIF4E protein phosphorylated at Ser209 directly in your cell type of interest. EIF4E enables the expression of key genes involved in cellular proliferation, angiogenesis, and cell survival. EIF4E modulation has applications in metabolism/diabetes, neurosciences and oncology research.

Benefits

  • VALIDATED ON ENDOGENOUS EXPRESSION
  • SPECIFICITY

Phospho-EIF4E (Ser209) assay principle

The Phospho-EIF4E (Ser209) assay measures EIF4E when phosphorylated at Ser209. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-EIF4E (Ser209) 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-EIF4E (Ser209) assay principle

Phospho-EIF4E (Ser209) 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-EIF4E (Ser209) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-EIF4E (Ser209) 2-plate assay protocol

Phospho-EIF4E (Ser209) 1-plate assay protocol

Detection of Phosphorylated EIF4E (Ser209) 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-EIF4E (Ser209) 1-plate assay protocol

Western Blot versus HTRF assay

HEK293 cells were cultured for 1 day in a T175 flask until 80% confluency was reached.. Following lysis and centrifugation, equal amounts of lysates in serial dilutions were analyzed in a side by side comparison of Western Blot and HTRF.. The HTRF assay is determined to be 8-fold more sensitive than Western Blot.
HTRF Phoshpo-EIF4E (Ser209) assay compared to Western Blot

Dose-response of CGP57380 on NIH3T3 cells

Murine NIH3T3 cells (50,000 cells/well) were incubated for 3 hours with various concentrations of CGP57380 inhibitor. After a 30 minutes lysis incubation, inhibition of eIF4E phosphorylation was measured using the HTRF phospho-eIF4E (Ser209) assay with the two-plate protocol.
Dose-response of CGP57380 on NIH3T3 cells

Dose-response of CGP57380 on stimulated HeLa cells

Human HeLa cells (100,000 cells/well) were incubated with various concentrations of CGP57380 inhibitor, either for 30 minutess or 3 hours, followed by stimulation with 10 µM Anisomycin for 30 minutess at 37°C. After a 30 minutes lysis incubation, inhibition of eIF4E phosphorylation was measured using the HTRF phospho-eIF4E (Ser209) assay with the two-plate protocol.
Dose-response of CGP57380 on stimulated HeLa cells

Simplified Pathway for EIF4E

EIF4E, eukaryotic translation initiation factor 4E, initiates eukaryotic protein synthesis by binding to the 5’ end of the mRNA cap structure and directing its association with the ribosomes. EIF4E enables the expression of key genes such as cyclins, c-myc, and vascular endothelial growth factor, involved in cell proliferation, angiogenesis and cell survival. Overexpression of phosphorylated eIF4E at Ser209 is associated with tumorgenesis, metastasis, and poor overall survival in several cancers making it a novel research target.
EIF4E signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

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

Automation of HTRF® elF4E Kinase Assay Using a 3D Tumoroid-Based cell Model

MultiFlo™ FX Microplate Dispenser (BioTek) - 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

p-S6RP, p-p70S6K and p-EIF4E - Validation of three new HTRF® phospho-assays for the study of translational control.

p-S6RP, p-p70S6K and p-EIF4E - Validation of three new HTRF® phospho-assays for the study of translational control. - Posters

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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