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Total-VEGFR2 cellular kit HTRF®

The Total VEGFR2 kit monitors the cellular VEGFR2 expression level and can be used as a normalization assay for the Phospho-VEGFR2 kit.

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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 Total VEGFR2 kit monitors the cellular VEGFR2 expression level and can be used as a normalization assay for the Phospho-VEGFR2 kit.

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Overview

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

Benefits

  • VALIDATED ON MULTIPLE CELL TYPES
  • SPECIFICITY

Total VEGFR2 assay principle

The total VEGFR2 assay measures VEGFR2. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The total VEGFR2 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.
Total FGFR2 assay principle

Total VEGFR2 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 VEGFR2 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total FGFR2 2-plate assay protocol

Total VEGFR2 1-plate assay protocol

Detection of total 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.
Total FGFR2 1-plate assay protocol

Insulin dose response effect on Phospho and Total VEGFR2 assays on Huvec cells

Huvec cells were plated at 150,000 cells per well in a 96-well plate. After an overnight incubation at 37°C, 5% CO2, a serial dilution of human VEGF was added to the cells for 3 minutes at 37°C, 5% CO2. Stimulation medium was removed from cells and 50µL of lysis buffer were added. A lysis step was carried out, shaking gently for 30 minutes. 16µL of samples were transferred into a 384-well small volume plate, then 4µL of Phospho VEGFR2 HTRF detection reagents were added. In parallel, 16µl were dispensed into other wells, then 4µL of HTRF Total VEGFR2 detection reagents were added. Signals were recorded overnight.

Human VEGF stimulation on Huvec cells

Western Blot comparison with Total VEGFR2 detection

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 into a 384-well low volume white microplate before finally adding VEGFR2 cellular kit detection reagents.

A side by side comparison showed the HTRF total assay is at least 32-fold more sensitive than the Western Blot.

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

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

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - 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

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

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

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

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 total kit / 64VEGFR2TPEG-64VEGFR2TPEH

64VEGFR2TPEG-64VEGFR2TPEH - Product Insert

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

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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