Phospho-VEGFR2 (Tyr1175) Cellular Assay Kit

Homogeneous cell-based assay for VEGF-mediated signaling in angiogenesis, vascular permeability, and regulation of cancer stem cells

Cisbio's cell-based homogeneous HTRF® phospho-VEGFR2 assay Tyr1175 enables quantitative detection of the modulation of the VEGF receptor 2, phosphorylated on Tyrosine 1175. This receptor binds the vascular endothelial growth factor VEGF, which makes VEGFR the key receptor in VEGF signaling regulation in angiogenesis, vascular permeability, tumorigenesis, cancer stem cells and tumorigenesis. Once VEGF receptor 2 is activated, mainly in the vascular endothelium, several pathways can be triggered, such as the PI3K/AKT pathway, the MAPK pathway or the focal adhesion pathway. These are involved mainly in angiogenesis and vasculogenesis, and thus play an important role in cancer research. With no wash steps and a simple add-and-read protocol, phospho-VEGFR2 assay serves as an ideal screening tool for small molecule compounds or biologics directly in cells, and offers enhanced convenience over Western Blot or ELISA.

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Assay Principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio's phospho-VEGFR2 assay is based on a TR-FRET sandwich immunoassay format comprising two specific anti-VEGFR2 antibodies, one labeled with a cryptate as donor and the other with d2 as acceptor. The phospho-VEGFR2 antibodies bind the phosphorylated residue, and the proximity of donor and acceptor will then lead to a fluorescent TR-FRET signal. The signal intensity is proportional to the substrate phosphorylation. 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 phospho-VEGFR2 assay kit can be run with frozen cell lysates or fresh cells in culture. After cell lysis, phospho-VEGFR2 can be quantitatively detected using the HTRF phospho-VEGFR2 kit reagents and most TR-FRET multimode plate readers.

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 the detection of phosphorylated VEGFR2, lysates are subsequently transferred to a 384 small volume assay plate where the HTRF reagents are added. This also enables the monitoring of cell viability and confluence in an appropriate cell culture plate.

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.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
VEGFR phospho-Y1175 kit - 500 tests63ADK041PEG
VEGFR phospho-Y1175 kit - 10,000 tests63ADK041PEH
VEGFR phospho-Y1175 kit - 50,000 tests63ADK041PEY
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