The total HER3 kit monitors the cellular receptor expression level and can be used as a normalization assay for the phospho-HER3 kit.
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  • High sensitivity High sensitivity
  • Long stability Long stability
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
The total HER3 kit monitors the cellular receptor expression level and can be used as a normalization assay for the phospho-HER3 kit.
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Overview

The Total HER3 cellular assay monitors HER3 and is used as a normalization assay with the phospho-HER3 kit. Overexpression of HER3, or human epidermoid receptor 3/ ErbB3, is considered as an indicator for a poor prognosis in human breast cancer. Heterodimerization of HER3 with HER2 is associated with an increase in the aggressiveness of a breast cancer. HER3 has also been found to be involved in prostate, gastric, colon, or other carcinomas. This makes HER3 a key target for anti-cancer therapies.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total-HER3 assay principle

The Total-HER3 assay quantifies the expression level of HER3 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-HER3 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 HER3 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 HER3 cellular assay principle

Total-HER3 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 HER3 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total HER3 2-plate assay protocol

Total-HER3 1-plate assay protocol

Detection of total HER3 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 HER3 1-plate assay protocol

Phospho-HER3 inhibition by Tyrosine kinase inhibitors and mAbs

100,000 cells were cultured prior to pre-treatment with a dose-response of inhibitors. The cells were or were not stimulated with 100 nM HRG Beta1 at 37 °C, 5% CO2. After these steps, medium was removed and cells were lysed with 50 µL of Lysis buffer for 30 min at RT under gentle shaking. For detection, 16 µL of lysate were transferred into 384-well sv white microplates: 4 µL of the HTRF phospho-HER3 detection reagents were added to detect the phosphorylated HER3 and 4 µL of the HTRF total-HER3 assay to detect the total protein. The HTRF signal was recorded after an overnight incubation.
Phospho-HER3 inhibition by Tyrosine kinase inhibitors and AB's

Intracellular inhibition by tyrosine kinase inhibitor Lapatinib and normalization by Total HER3 quanification

Human breast carcinoma MDA-MB-453 and SKBR3 cells were pre-treated for 30 min with a dose-response of Lapatinib, a small Tyrosine Kinase Inhibitor, then stimulated for 10 min with 100 nM HRG Beta1 for 10 min.
Intracellular inhibition by Lapatinib, a Tyrosine Kinase Inhibitor
Intracellular inhibition by Lapatinib, a Tyrosine Kinase Inhibitor

Inhibition by mAbs and normalization by Total HER3 quanification

Human breast carcinoma BXPC3 cells were pre-treated for 30 min with a dose-response of mAb-X or mAb-Y, then stimulated for 10 min with 100 nM HRG Beta1 for 10 min. Phosphorylation modulation was normalized vs total HER3. Both mAbs were kindly provided by Dr Thierry Chardès (IRCM Montpellier, France).
Inhibition of phospho-HER3 by monoclonal antibodies
Inhibition by mAb-X and mAb-Y kindly provided by Dr Thierry Chardès

HER3 Epidermal growth factor signaling

HER3 is a receptor tyrosine kinase and belongs to the ErbB family of epidermal growth factor receptors. HER3 is present on the cell surface and is the only EGFR family member for which no ligand has been found yet. HER3 receptor activation induces auto-phosphorylation of HER3 on Tyr1289. This provides docking sites for a variety of adaptor proteins, kinases & phosphatases that induce downstream activation of several signal transduction cascades. The HER3/ErbB receptor regulates various biological processes such as cell proliferation, differentiation, survival, adhesion, migration & angiogenesis. Hyperactivity of HER3 is associated with cancer which make HER3 a key target for anti-cancer therapies. Heterodimerization of HER3 with HER2 relates to an increase in cancer aggressiveness.
HER3 Epidermal growth factor 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

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

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 HER3 total Kit / 64NR3PEG-64NR3PEH

64NR3PEG-64NR3PEH - 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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