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HTRF Human Phospho-HER2 (Tyr1221/1222) Detection Kit, 10,000 Assay Points

Her2 P-y1221/1222 Kit - 50,000 Tests

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).
Part Number: 64HR2PEG
Unit Size: 500 Assay Points
Part Number: 64HR2PEH
Unit Size: 10,000 Assay Points
  • 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.

  • Assay Points
    10000
    Assay Target Type
    Kit
    Assay Technology
    HTRF
    Brand
    HTRF
    Quantity
    1
    Therapeutic Area
    Oncology & Inflammation
    Unit Size
    10,000 Assay Points
  • 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.

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    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.

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    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.

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  • 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.

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    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.

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    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.

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  • 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.

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Guide
HTRF solutions, guide to major applications

This guide provides you an overview of HTRF applications in several therapeutic areas.