VEGFR1 domain 2 covalent labeling with horseradish peroxidase: Development of a displacement assay on VEGF

The VEGFR1 has been shown to play a role in the regulation of angiogenesis, and has therefore been associated to several pathologies. In order to extend our toolbox of screening methods for the identification of compounds disrupting the VEGF receptor 1/VEGF interaction, we developed a fast and accur...

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Bibliographic Details
Published in:Analytical biochemistry 2017-08, Vol.530, p.107-112
Main Authors: Reille-Seroussi, Marie, Gaucher, Jean-François, Cussac, Laure-Anne, Broutin, Isabelle, Vidal, Michel, Broussy, Sylvain
Format: Article
Language:English
Subjects:
Angiogenesis
Binding, Competitive
Biological Assay - methods
Biotin - chemistry
Biotin - metabolism
Covalent labeling
Displacement assay
Enzyme-Linked Immunosorbent Assay
Horseradish Peroxidase - chemistry
Horseradish Peroxidase - metabolism
HRP
Humans
Life Sciences
Ligands
Peptide Fragments - chemistry
Peptide Fragments - metabolism
Protein Binding
Streptavidin - chemistry
Streptavidin - metabolism
Vascular Endothelial Growth Factor A - chemistry
Vascular Endothelial Growth Factor A - metabolism
Vascular Endothelial Growth Factor Receptor-1 - chemistry
Vascular Endothelial Growth Factor Receptor-1 - metabolism
VEGF
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Summary:The VEGFR1 has been shown to play a role in the regulation of angiogenesis, and has therefore been associated to several pathologies. In order to extend our toolbox of screening methods for the identification of compounds disrupting the VEGF receptor 1/VEGF interaction, we developed a fast and accurate displacement assay, in which VEGF receptor 1 domain 2 is directly labeled with an enzyme, bypassing the classical streptavidin-biotin interaction system. A description of this straightforward strategy is provided here, including its advantages and disadvantages. Optimization of the reagents preparation, purification and conservation, and displacement assay with known molecular entities are presented. •We produced, characterized and purified a covalent conjugate VEGFR1d2-HRP.•The VEGFR1d2-HRP conjugate retained a dissociation constant for VEGF of 15 nM.•A displacement assay was developed for both VEGFR1d2 and VEGF binding ligands.•The displacement assay was accurate and faster than a previously described assay.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2017.05.004