Phospho-EGFR (Tyr1068) cellular kit

The phospho-EGFR (Tyr1068) kit is designed for the cell-based quantitative detection of EGFR phosphorylation.
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  • Highly specific Highly specific
  • Ease-of-use Ease-of-use
The phospho-EGFR (Tyr1068) kit is designed for the cell-based quantitative detection of EGFR phosphorylation.
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Overview

The phospho-EGFR assay kit is designed for the cell-based quantitative detection of Tyr1068 phosphorylation 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.

Benefits

  • COMPATIBLE WITH MANY CELL TYPES
  • SPECTIFICITY

Phospho-EGFR (Tyr1068) assay principle

The Phospho-EGFR (Tyr1068) assay measures EGFR when phosphorylated at Tyr1068. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-EGFR (Tyr1068) 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 (Tyr1068) assay principle

Phospho-EGFR (Tyr1068) 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-EGFR (Tyr1068) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-EGFR (Tyr1068) 2-plate assay protocol

Phospho-EGFR (Tyr1068) 1-plate assay protocol

Detection of Phosphorylated EGFR (Tyr1068) 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-EGFR (Tyr1068) 1-plate protocol

HTRF phospho-EGFR (Tyr1068) assay compared to WB

Human A431 cells were cultured for 48 h and stimulated with 100 EGF for 10 min. Following lysis, soluble fractions were collected via centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were dispensed and analyzed side-by-side by Western-blot and by HTRF. By using HTRF phospho-EGFR (Tyr1068) only 100 cells are sufficient for minimal signal detection while 3,120 cells are needed for a Western Blot signal. The HTRF phospho-EGFR assay is at least 30-fold more sensitive than the Western Blot and shows optimal correlation.
HTRF phospho-EGFR (Tyr1068) assay compared to Western Blot
HTRF phospho-EGFR (Tyr1068) correlation with Western Blot

Phospho-EGFR modulation in several human tumor cell lines

Human BxPC3 (Figure 1;green), SKOV3 (Figure 2;purple), CRC1 (Figure 3) and A431 (Figure 4;pink) cells were plated in 96 well plate and incubated for 24 h, at 37 °C-5% Co2. After 10 min incubation with increasing concentrations of agonist, medium was removed and cells were lysed with 50 µL of Lysis buffer for 30min at RT under gentle shaking.16 µL of lysate was transfered into a 384 sv white microplate and 4 µL of the HTRF phospho-EGFR detection reagents were added. HTRF signal was recorded after an overnight incubation.
Figure 1: Increasing concentration of EGF added to human pancreatic carcinoma BxPC3 cells
Figure 2: Increasing concentration of TGF-a on human epidermoid carcinoma A431 cells
Figure 3: Increasing concentration of ß-cellulin on human ovarian carcinoma SKOV3 cells
Figure 4: Increasing concentration of EGF on CRC1, human colorectal primary tumour cells

Inhibition measured with Phospho (tyr1068) & total EGFR kits

Human epidermoid carcinoma A431 cells were pre-treated for 3 h with a dose-response of Cetuximab, an extracellular therapeutic antibody, then stimulated for 10 min with 200 nM EGF (Figure 1: Left). Human pancreatic carcinoma BxPC3 cells were pre-treated for 30 min with a dose-response of ATCC528, an extracellular monoclonal antibody, then stimulated for 10 min with 200 nM EGF (Figure 2: right).
Figure 1: Phospho-EGFR inhibition with Cetuximab
Figure 2: Phospho-EGFR inhibition with mAB ATCC528
Human epidermoid carcinoma A431 cells were pre-treated for 3h with a dose-response of Gefitinib, a small Tyrosine Kinase Inhibitor, then stimulated for 10 min with 200 nM EGF. As expected, results obtained show a dose-response decrease of EGFR phosphorylation upon treatment with inhibitors while EGFR expression level remains constant
Phospho-EGFR inhibition with Gefitinib, a small Tyrosine Kinase Inhibitor

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 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

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

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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