Phospho-HER3 (Tyr1289) & Total HER3 Cellular Assay Kits

HTRF® cell-based phospho and total-HER3 assays for anti-cancer research

Overexpression of HER3, 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 the increase in the aggressiveness of a breast cancer. HER3 is also been found to be involved in prostate, gastric, colon or other carcinomas. This makes HER3 a key target for anti-cancer therapies when screening or profiling small molecule TK inhibitors or biotherapeutic antibodies to induce ADCC and/or block HER3 heterodimerization.

Cisbio’s homogeneous and robust HTRF® phospho-HER3 (Tyr1289) assay kit is designed for the cell-based quantitative detection of HER3 signaling activity. The HTRF® total HER2 assay kit is designed for the quantitative detection of total HER3, phosphorylated and unphosphorylated, to normalize the phosphorylation status of HER3.

The buffers of both HTRF® phospho- and total HER3 assays are compatible, enabling an analysis of the phosphorylated and the total protein populations from one lysate sample.

   Check our lysis buffer compatibility      Check our species compatibility

Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total HER3 assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-HER3 antibodies, one labeled with Cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with HER3, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-HER3 can be quantitatively detected using the HTRF phospho-HER3 (Tyr1289) and total HER3 cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol – adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384-well small volume assay plate where the HTRF reagents are added. This also enables monitoring the cells' viability and confluence in an appropriate cell culture plate.

One-plate assay protocol

The protocol can be further streamlined to a one-plate assay in which plating, treating and detecting are all performed in a single plate. No wash steps are required. Ideal for HTS - this protocol means enhanced speed and simplicity, enabling all throughputs and fast results while maintaining high quality and sensitive output.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Simplified Pathway

HER3 Epidermal growth factor signaling

HER3, also known as ErbB3 or human epidermoid receptor 3, is a receptor tyrosine kinase and belongs to the ErbB/EGFR family of epidermal growth factor receptors. HER3 is expressed on the cell surface. The HER3/ErbB3 extracellular domain binds its natural ligand, Heregulin beta. The HER3 catalytic domain remains unfunctional. Homo- or heterodimerization with another ErbB/EGFR family receptor induces receptor activation and subsequently phosphorylation of HER3 on Tyr1289. The phosphorylated tyrosine residues then transduct the signal via a variety of adaptor proteins, kinases & phosphatases that induce downstream activation of several signal transduction cascades, principally the MAPK/ERK/JNK and PI3K/AKT pathways. Further inductions that may result from receptor activation are STAT signaling, Calcium/PKC signaling and nuclear localization of the receptor. The heterodimerization of HER3 to other HER/ErbB receptors results in increased signaling potency of the dimerized receptors through increased ligand affinity, increased coupling efficiency to signaling molecules, and a decreased rate of receptor internalization. The signal transduction from the HER3/ErbB receptor dimers to the nucleus via AKT and ERK results in the regulation of various biological processes such as cell proliferation, differentiation, survival, adhesion, migration & angiogenesis. Overexpression of HER3 is considered as an indicator for a poor prognosis in human breast cancer. Heterodimerization of HER3 with HER2 is associated with the increase in the aggressiveness of a breast cancer. HER3 is also been found to be involved in prostate, gastric, colon or other carcinomas. This  makes HER3 a key target for anti-cancer therapies when screening or profiling small molecule TK inhibitors or biotherapeutic antibodies to induce ADCC and/or block HER3 heterodimerization.

Product Performances

1. HTRF Phospho-HER3 assays compared to Western Blot

MDA-MB-453 cells were grown in a T175 flask at 37 °C, 5% CO2 for 48 h. Cells were then stimulated with 100 nM HRG Beta1 for 10 min. After medium removal, the cells were lysed with 3 mL of supplemented lysis buffer for 30 min at room temperature. Soluble fractions were collected after a 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.

Using HTRF phospho-HER3 (Tyr1289) only 5,000 cells are sufficient for minimal signal detection, while 80,000 cells are needed for a Western Blot signal. The HTRF phospho-HER3 assay is at least 16-fold more sensitive than the Western Blot.

2. Phospho HER3 modulation in several breast adenocarcinoma cell lines

100,000 human recombinant HER2/HER3, MCF-7, SK-BR-3 and MDA-MB-453 cells were plated in 96 well plates and incubated for 24h at 37 °C-5% Co2. After incubation with increasing concentrations of HRG Beta3 (10min), medium was removed and cells were lysed with 50µl of Lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate and 4 µL of the HTRF phospho-HER3 detection reagents were added. HTRF signal was recorded after an overnight incubation.

3. Inhibition of HER3 phosphoryation by Tyrosine kinase inhibitors and therapeutic antibodies

100,000 cells were plated in 96-well plate and incubated for 24 h at 37 °C - 5% Co2. Then, after a 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 Lapatinib, a Tyrosine Kinase Inhibitor

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.

 

 

Inhibition by mAb-X and mAb-Y, two monoclonal antibodies kindly provided by Dr Thierry Chardès, IRCM Montpellier

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.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
HER3 phospho-Y1289 kit - 500 tests64HR3PEG
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HER3 phospho-Y1289 kit - 10,000 tests64HR3PEH
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HER3 phospho-Y1289 kit - 50,000 tests64HR3PEY
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HER3 total kit - 500 tests64NR3PEG
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HER3 total kit - 50,000 tests64NR3PEY
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Companion products

DescriptionCat. noProduct insertMSDS
HER3 phospho-Y1289 kit control lysate64HR3TDA
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HER3 total kit - 10,000 tests64NR3PEH
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HER3 total kit control lysate64NR3TDA
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