The total HER2 kit monitors the cellular receptor expression level and can be used as a normalization assay for the phospho-HER2 kit.
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  • Ease-of-use Ease-of-use
  • High sensitivity High sensitivity
  • Highly specific Highly specific
The total HER2 kit monitors the cellular receptor expression level and can be used as a normalization assay for the phospho-HER2 kit.
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Overview

The Total HER2 cellular assay kit monitors HER2 and is used as a normalization assay with the phospho-HER2 kit. 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
  • DATA NORMALIZATION

Total-HER2 assay principle

The Total-HER2 assay quantifies the expression level of HER2 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-HER2 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 HER2 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 HER2 cellular assay principle

Total-HER2 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 HER2 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total HER2 2-plate assay protocol

Total-HER2 1-plate assay protocol

Detection of total HER2 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 HER2 1-plate assay protocol

HTRF Total HER2 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 3mL 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 total-HER2, only 3000 cells are sufficient for minimal signal detection while 12,500 cells are needed for a Western Blot signal. The HTRF assay is at least 4-fold more sensitive than the Western Blot.
HTRF total HER2 assay vs Western Blot

Validation of the HTRF total-HER2 assay on human cancer cell lines

Human (SKOV3, BT474, A431 and MCF7) cells were grown in a T175 flask at 37 °C, 5% CO2 for 2 days. After removal of cell culture medium, 3 mL of supplemented lysis buffer 3 were added and incubated for 45 min. Soluble supernatants were collected after 10 min centrifuging. HER2 was detected using 16µL of cell lysate. Cell lines from other species have not been tested, hence they must be evaluated case by case.the Western Blot. Differences obtained for S/N are well correlated with the number of HER2 receptors present in the different cell lines (Gabott et all., JBC 2011)
Validation of the HTRF total-HER2 assay on human cancer cell lines

Total HER2 assay used to control the phosphorylation status of HER2

SKOV3 cells (100,000 cells/well) were activated with EGF for 10 min, using the two-plate assay protocol of the HTRF phospho- and total-HER2 cellular assays. As expected, results obtained show a dose-response increase of HER2 phosphorylation upon EGF stimulation while HER2 expression level remains constant.
Total HER2 assay used to control the phosphorylation status of HER2 - results

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 make 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

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 HER2 total Kit / 64NR2PEG-64NR2PEH

64NR2PEG-64NR2PEH - 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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