Pan-phospho-EGFR cellular kit HTRF®

The pan phospho-EGFR kit is designed to monitor all tyrosine phosphorylation on EGFR.
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The pan phospho-EGFR kit is designed to monitor all tyrosine phosphorylation on EGFR.


The pan-phospho-EGFR assay is designed for the cell-based quantitative detection of all tyrosine phosphorylations on EGFR. Mutations that lead to an overexpression or hyperactivity of EGFR, also known as ErbB1 or HER1, are associated with a number of cancers, such as lung, colorectal, prostate, glioblastoma cancer, etc. This makes EGFR a key target for anti-cancer therapy-related immunoassay.



Phospho-EGFR (pan) assay principle

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

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

Phospho-EGFR (pan) 1-plate assay protocol

Detection of Phosphorylated Pan EGFR 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.
Pan Phospho-EGFR 1-plate protocol

EGF dose-response on A431 cells

Human A431 cells (5,000 cells/well) were stimulated for 30 min at 37°C with various concentrations of EGF. After a 30 min lysis incubation time, phosphorylated EGFR was measured using the one-plate assay protocol of the pan-EGFR assay.
EGF dose-response on A431 cells and subsequent measurement of phosphorylated Erk

Inhibition of EGFR phosphorylation

Cells were plated in 96-well plate and incubated for 24h. After a pre-treatment with a dose-response of inhibitors, the cells were stimulated with EGF. Following cell lysis, lysates were transferred into a 384-sv white microplate for detection and HTRF phospho-EGFR detection reagents were added. HTRF signal was recorded after an overnight incubation. Results show the applicability of the HTRF phospho-EGFR assay to decipher the mechanism of action of small molecules and biologics targeting EGFR.
Inhibition of EGFR phosphorylation

Inhibition of EGFR phosphorylation

Cells were pre-treated with a dose-response of Erlotinib and Lapatinib, 2 small Tyrosine Kinase inhibitors. compound. A control was done using PQ401, a non-inhibitor compound (Figure 1 on the left). Cells were pre-treated with a dose-response of Cetuximab, a therapeutic monoclonal antibody. A control was done using a non-inhibitor MAb control (15441) (Figure 2 on the right).
Inhibition of EGFR phosphorylation (Figure 1)
Inhibition of EGFR phosphorylation (Figure 2)

EGFR Epidermal growth factor receptor signaling Simplified Pathway

EGFR is a receptor tyrosine kinase that belongs to the ErbB family. Binding of EGFR ligands drive receptor homodimerization or hetero-dimerization leading to the activation of the EGFR tyrosine kinase domain and specific tyrosine residues. The phosphorylated tyrosine residues then provide docking sites for a variety of factors that induce downstream activation of several signal transduction cascades. The transmitted signals from the EGF receptor to the nucleus lead to the regulation of various biological functions such as cell proliferation, differentiation, survival, adhesion, migration & angiogenesis. Mutations that lead to overexpression or hyperactivity of EGFR are associated with a number of cancers making EGFR a key target for anti-cancer therapies.
EGFR 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

Lysis buffer compatibility

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

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Species compatibility

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

Reagent solutions for PROTAC research

Our offer for PROTAC research summarized in this flyer - Flyers

PROTAC assays design at-a-glance

Streamlined set-up and access to multiple read-out options for research in oncology - Application Notes

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

HTRF Product Catalog

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

Product Insert EGFR P-pan Kit / 64HR1PEG-64HR1PEH

64HR1PEG-64HR1PEH - Product Insert

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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