The pan phospho-EGFR kit is designed to monitor all tyrosine phosphorylation on EGFR.
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  • Ease-of-use
  • High sensitivity
The pan phospho-EGFR kit is designed to monitor all tyrosine phosphorylation on EGFR.
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Overview

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.

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.

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.

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.

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

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).

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.
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

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

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

Deciphering the mechanism of action of drugs targeting EGFR, by Cisbio’s advanced cell-based pathway readout assays.

mechanism of action of EGFR inhibitors assessed in the pancreatic BXPC3 cell line - 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

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