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Phospho-VEGFR2 (Tyr1175) cellular kit HTRF®

The phospho-VEGFR2 (Tyr1175) kit is designed to enable quantitative detection of the modulation of the VEGF receptor 2, phosphorylated on Tyrosine 1175.
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  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
  • No-wash No-wash
The phospho-VEGFR2 (Tyr1175) kit is designed to enable quantitative detection of the modulation of the VEGF receptor 2, phosphorylated on Tyrosine 1175.
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Overview

Homogeneous cell-based assay for monitoring phospho-VEGFR2 (Tyr1175) modulations. VEGFR2 mediated signaling data has uses in angiogenesis, vascular permeability and cancer stem cell regulation research.

Benefits

  • VALIDATED ON MULTIPLE CELL TYPES
  • SPECIFICITY

Phospho-VEGFR2 (Tyr1175) assay principle

The Phospho-VEGFR2 (Tyr1175) assay measures VEGFR2 when phosphorylated at Tyr1175. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-VEGFR2 (Tyr1175) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize 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.
Phospho-VEGFR2 (Tyr1175) assay principle

Phospho-VEGFR2 (Tyr1175) 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-VEGFR2 (Tyr1175)HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-VEGFR2 (Tyr1175) 2-plate assay protocol

Phospho-VEGFR2 (Tyr1175) 1-plate assay protocol

Detection of phosphorylated VEGFR2 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.
Phospho-VEGFR2 (Tyr1175) 1-plate assay protocol

Human VEGF stimulation on Huvec cells leads phosphorylation on VEGFR2 protein on tyrosine 1175 residue

HUVEC cells were plated at various cellular densities on a 96-well plate. After overnight incubation at 37°C and 5% CO2, a serial dilution of human VEGF was added for 3 minutes at 37°C and 5% CO2. Stimulation medium was removed from cells and 50µL of lysis buffer was added onto the cells. The lysis step was done by shaking gently for 30 minutes. 16µL of samples were transferred to a 384-well small volume plate, then 4µL of Phospho VEGFR2 HTRF detection reagents were added. Signals were recorded overnight. The assay window increased as cellular density increased.
Human VEGF stimulation on Huvec cells leads phosphorylation on VEGFR2 protein on tyrosine 1175 residue

Stimulation kinetic of VEGF on phospho-VEGFR2 Tyr1175 cellular assay

HUVEC cells were plated at 100,000 cells/well on a 96-well plate. After overnight incubation at 37°C and 5% CO2, a serial dilution of human VEGF was added for various times at 37°C and 5% CO2. Stimulation medium was removed from cells and 50µL of lysis buffer was added onto the cells. The lysis step was done by shaking gently for 30 minutes. 16µL of samples were transferred to a 384-well small volume plate, then 4µL of Phospho VEGFR2 HTRF detection reagents were added. Signals were recorded overnight. Short stimulation time is more effective for an optimal assay window.
Stimulation kinetic of VEGF on phospho-VEGFR2 Tyr1175 cellular assay

HTRF assay compared to Western Blot using phospho-VEGFR2 cellular assay on Huvec cells

Human HUVEC cells were cultured to 80% confluency. After 3 minutes of hVEGF treatment, cells were lysed and soluble supernatants were collected via centrifugation. Serial dilutions of the cell lysate were performed and 16 µL of each dilution were transferred to a 384-well low-volume white microplate before finally adding VEGFR2 cellular kit detection reagents. A side-by-side comparison showed that the HTRF total assay is more sensitive than Western Blot by a factor of at least 32.
HTRF assay compared to Western Blot using phospho-VEGFR2 cellular assay on Huvec cells

VEGFR2 signaling pathway

The vascular endothelial growth factor (VEGF) is an endothelial-specific mitogen factor that is often associated with the activation of tumour-induced angiogenesis. In tumours, VEGF secretion is upregulated. At the endothelial cell surface, the secreted protein binds with high affinity to at least two distinct vascular endothelial growth factor receptors (VEGFRs), VEGFR-1, also known as fms-like tyrosine kinase (Flt-1) and VEGFR-2, also known as fetal liver kinase (Flk-1). Activation of endothelial cells by VEGF leads to the receptor auto-phosphorylation and subsequent signaling. Multiple phosphorylation sites have been described. VEGFR is implicated in angiogenesis, and combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression
VEGFR2 signaling pathway

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

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

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

HTRF Product Catalog

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

Product Insert VEGFR2 P-Y1175 Kit / 63ADK041PEG-63ADK041PEH

63ADK041PEG-63ADK041PEH - Product Insert

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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