Total EGFR cellular kit

The total EGFR kit monitors cellular EGFR expression level and can be used as a normalization assay for the phospho-EGFR kits.
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The total EGFR kit monitors cellular EGFR expression level and can be used as a normalization assay for the phospho-EGFR kits.
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Overview

The Total EGFR cellular assay kit monitors total EGFR and is used as a normalization assay with our phospho-EGFR kits. 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

  • SPECIFICITY
  • COMPATIBLE WITH MANY CELL TYPES
  • DATA NORMALIZATION

Total-EGFR assay principle

The Total-EGFR assay quantifies the expression level of EGFR in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-EGFR assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of EGFR in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
Total EGFR assay principle

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

Total-EGFR 1-plate assay protocol

Detection of total 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.
Total EGFR 1-plate protocol

HTRF total EGFR assay compared to WB

A431 cells were grown in a T175 flask at 37 °C, 5% CO2 for 48h. Cells were then stimulated with 100 nM EGF for 10 min. After medium removal, the cells were lysed with 3mL of supplemented lysis buffer for 30 min at room temperature. Soluble fractions were then collected after 10 min 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 total-EGFR only 50 cells are sufficient for minimal signal detection while 780 cells are needed for a Western Blot signal. The HTRF total-EGFR assay is at least 15-fold more sensitive than the Western Blot and shows optimal correlation.
HTRF total EGFR assay compared to Western Blot
Correlation between Wester Blot and HTRF total EGFR assays

Inhibition measured with Phospho-EGFR (tyr1068) and total EGFR cellular 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.
Cetuximab inhibition of EGFR phosphorylation

Inhibition measured with Phospho-EGFR (Tyr1068) and total EGFR cellular kits

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
mAB ATCC528 inhibition of EGFR phosphorylation

Inhibition measured with Phospho-EGFR (tyr1068) and total EGFR cellular kits

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
Gefitinib inhibition of EGFR phosphorylation

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

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

Product Insert EGFR total Kit / 64NG1PEG-64NG1PEH

64NG1PEG-64NG1PEH - Product Insert

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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