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HTRF Human Total ErbB2 (HER2) Detection Kit, 500 Assay Points

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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Feature	Specification
Application	細胞シグナル伝達
Sample Volume	16 µL

The total HER2 kit monitors the cellular receptor expression level and can be used as a normalization assay for the phospho-HER2 kit.

View product information

Product variants

Unit Size: 500 assay points

Part #:

64NR2PEG

Unit Size: 10,000 assay points

Part #:

64NR2PEH

Unit Size: 50,000 assay points

Part #:

64NR2PEY

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, and disposal requirements under European REACH regulations (EC 1907/2006).

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Product information

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.

HTRF assays offer many advantages over other technologies:

Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays

How it works

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 proteins expression under a no-wash assay format.

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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 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.

Assay validation

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.

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)

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.

Simplified pathway

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.

phospho-pathway-her2-epidermal-growth-factor-64nr2peg

Specifications

Other specifications

Application	Cell Signaling
Brand	HTRF
Detection Modality	HTRF
Lysis Buffer Compatibility	Lysis Buffer 3 Lysis Buffer 4 Lysis Buffer 5
Molecular Modification	Total
Product Group	Kit
Sample Volume	16 µL
Shipping Conditions	Shipped in Dry Ice
Target	ErbB2 (HER2)
Target Class	Phosphoproteins
Target Species	Human
Technology	TR-FRET
Therapeutic Area	Oncology & Inflammation
Unit Size	500 assay points