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HTRF Human Total HER3 Detection Kit, 500 Assay Points
The total HER3 kit monitors the cellular receptor expression level and can be used as a normalization assay for the phospho-HER3 kit.
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption & disposal requirements under European REACH regulations (EC 1907/2006).
Product Variants
Product information
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.
Specifications
| Assay Points |
500
|
|---|---|
| Assay Target Type |
Kit
|
| Assay Technology |
HTRF
|
| Brand |
HTRF
|
| Quantity |
1
|
| Therapeutic Area |
Oncology & Inflammation
|
| Unit Size |
500 Assay Points
|
Video gallery
How it works
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 proteins expression under a no-wash assay format.
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 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.
Assay validation
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.
Intracellular inhibition by tyrosine kinase inhibitor Lapatinib and normalization by Total HER3 quantification
Human breast carcinoma 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.
Inhibition by mAbs and normalization by Total HER3 quantification
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).
Simplified pathway
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.
Resources
Guide
HTRF solutions, guide to major applications
This guide provides you an overview of HTRF applications in several therapeutic areas.
SDS, COAs, Manuals and more
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