Phospho-HER2 (Tyr1221/1222) & Total HER2 Cellular Assay Kits

HTRF® cell-based phospho and total-HER2 assays for anti-cancer research

Upregulation of HER2, 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, which makes HER2 a key target for anti-cancer therapies when screening or profiling small molecule tyrosine kinase inhibitors or biotherapeutic antibodies to induce ADCC or to block HER2 heterodimerization.

Cisbio’s homogeneous and robust HTRF® phospho-HER2 (Tyr1221/1222) assay kit is designed for the cell-based quantitative detection of HER2 signaling activity. The HTRF® total HER2 assay kit is designed for the quantitative detection of total HER2, phosphorylated and unphosphorylated, to normalize the phosphorylation status of HER2.

The buffers of both HTRF® phospho- and total HER2 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 HER2 assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-HER2 antibodies, one labeled with Cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with HER2, 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-HER2 can be quantitatively detected using the HTRF phospho-HER2 (Tyr1221/1222) and total HER2 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

HER2 Epidermal growth factor signaling

HER2, also known as ErbB2 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. The HER2 extracellular domain displays an extended-open conformation, making it the preferred dimerization partner of all HER/ErbB receptors. Upon receptor-ligand binding of growth factor stimuli, e.g. EGF or TGF-α, to EGFR, HER3/ErbB3, or HER4/ErbB4, the activation of their receptor tyrosine kinase domains is induced which leads subsequently to auto-phosphorylation and to homo- or heterodimerization with e.g. HER2. HER2 receptor activation induces then auto-phosphorylation of HER2 on Tyr1221/1222. The phosphorylated tyrosine residues in consequence provide docking sites for a variety of adaptor proteins, kinases & phosphatases, such as Shc, Grb2, PLCɣ & PI3K 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 HER2 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 HER2/ErbB receptor dimers via AKT and ERK to the nucleus results in the regulation of various biological processes such as cell proliferation, differentiation, survival, adhesion, migration & angiogenesis. Upregulation of HER2 activity results in increased signaling potency of the dimerized receptors, increased ligand affinity and coupling efficiency to signaling molecules, or in a decreased rate of receptor internalization.  Therefore, hyperactivity of HER2 is associated with cancer, mainly human breast cancer and several others, such as ovarian, stomach, bladder, salivary or lung carcinomas, which make HER2 a key target for anti-cancer therapies.

Product performances

1. HTRF total and phospho-HER2 (Tyr1221/1222) assays 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 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.

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 cell based phospho HER2 WB vs HTRF graph

 

HTRF cell based phospho HER2 product performance graph

 

HTRF cell based total HER2 WB vs HTRF graph

 

2. 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.

3. Inhibition of HER2 phosphorylation by Tyrosine kinase inhibitors and monoclonal antibodies

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

4. 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)

5. HTRF® 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.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
HER2 phospho-Y1221/1222 kit - 500 tests64HR2PEG
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HER2 phospho-Y1221/1222 kit - 10,000 tests64HR2PEH
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HER2 phospho-Y1221/1222 kit - 50,000 tests64HR2PEY
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HER2 total kit - 500 tests64NR2PEG
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HER2 total kit - 10,000 tests64NR2PEH
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HER2 total kit - 50,000 tests64NR2PEY
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Companion products

DescriptionCat. noProduct insertMSDS
HER2 phospho-Y1221/1222 kit control lysate64HR2TDA
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HER2 total kit control lysate64NR2TDA
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