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Phospho-HER2 (Tyr1221/1222) cellular kit HTRF®

The phospho-HER2 (Tyr1221/1222) kit enables the cell-based quantitative detection of human epidermoid growth factor 2 (HER2, Erbb2) signaling activity.

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  • Ease-of-use Ease-of-use
  • High sensitivity High sensitivity

The phospho-HER2 (Tyr1221/1222) kit enables the cell-based quantitative detection of human epidermoid growth factor 2 (HER2, Erbb2) signaling activity.

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Overview

The phospho-HER2 (Tyr1221/1222) assay is designed for the robust quantification of HER2-Erbb2 modulation when phosphorylated on Tyr1221/1222, an indicator of many types of cancer pathologies. Upregulation of HER2, or human epidermoid receptor 2, also known as ErbB2 receptor, is associated with human breast cancer and several others, such as ovarian, stomach, bladder, salivary, or lung carcinomas, making HER2 a key target for anti-cancer therapies.

Benefits

  • SPECIFICITY
  • PRECISION

Phospho-HER2 (Tyr1221/1222) assay principle

The Phospho-HER2 (Tyr1221/1222) assay measures HER2 when phosphorylated at Tyr1221/1222. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-HER2 (Tyr1221/1222) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody specifically binds to the phosphorylated motif on the protein, the second recognizes 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-HER2 (Tyr1221/1222) cellular assay principle

Phospho-HER2 (Tyr1221/1222) 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-HER2 (Tyr1221/1222) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-HER2 (Tyr1221/1222) 2-plate assay protocol

Phospho-HER2 (Tyr1221/1222) 1-plate assay protocol

Detection of Phosphorylated HER2 (Tyr1221/1222) 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-HER2 (Tyr1221/1222) 1-plate assay protocol

HTRF phospho-HER2 (Tyr1221/1222) assay compared to Western Blot

Human SKOV3 cells were grown in a T175 flask at 37°C until 80% confluency. Cells were then stimulated with 100 nM EGF for 10min. After medium removal, the cells were lysed with 3 mL of supplemented lysis buffer for 30 min at room temperature. Soluble supernatants were collected after a 10 minute 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-HER2 (Tyr 1221/1222), only 1500 cells are sufficient for minimal signal detection while 12,500 cells are needed for a Western Blot signal. The HTRF assay is at least 8-fold more sensitive than the Western Blot and shows optimal correlation.
HTRF phospho-HER2 (Tyr1221/1222) assay compared to Western Blot
Sensitivity comparison of HTRF phospho-HER2 (Tyr1221/1222) assay vs Western Blot

Phospho-HER2 modulation in several human tumor cell lines

100,000 Human SKOV3, SK-BR-3, A431 and MCF-7 cells were plated in 96 well plate and incubated for 24h, at 37°C-5%Co2. After incubation with increasing concentrations of mEGF (10min), medium was removed and cells were lysed with 50µl of Lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate was transfered into a 384-well sv white microplate and 4 µL of the HTRF phospho-HER2 detection reagents were added. HTRF signal was recorded after an Overnight incubation.
Phospho-HER2 modulation in several human tumor cell lines

phospho-HER2 Inhibition by Tyrosine kinase inhibitors and mAbs

Experiments were run on SKOV3 cells. 100,000 cells were plated in 96-well plate and incubated for 24h, at 37°C-5%Co2, then, after a pre-treatment with a dose-response of inhibitors: Trastuzamab, Cetuximab and Pertuzumab (Therapeutic monoclonal antibodies) and Lapatinib (Tyrosine Kinase Inhibitor), the cells were stimulated with mEGF for 10 min at 37 °C, 5% CO2. After these steps, 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 transferred into a 384-sv white microplate for detection and 4 µL of the HTRF phospho-HER2 detection reagents were added. HTRF signal was recorded after an Overnight incubation. Results show the applicability of the HTRF phospho-HER2 assay to decipher the mechanism of action of small molecules and biologics targeting HER2.
phospho-HER2 Inhibition by Tyrosine kinase inhibitors and mAB's
phospho-HER2 Inhibition by mAbs
phospho-HER2 Inhibition by tyrosine kinase inhibitor lapatinib

HER2 Epidermal growth factor signaling

HER2 is a receptor tyrosine kinase and belongs to the ErbB family of epidermal growth factor receptors. HER2 is present on the cell surface and is the only EGFR family member for which no ligand has been found yet. HER2 receptor activation induces auto-phosphorylation of HER2 on Tyr1221/1222. This provides docking sites for 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. The signal transduction from the HER2/ErbB receptor regulates various biological processes such as cell proliferation, differentiation, survival, adhesion, migration & angiogenesis. Hyperactivity of HER2 is associated with cancer which makes HER2 a key target for anti-cancer therapies.
HER2 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

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

HTRF Product Catalog

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

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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