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

Anal Biochem. 2017 Aug 1:530:107-112. doi: 10.1016/j.ab.2017.05.004. Epub 2017 May 3.

Abstract

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.

Keywords: Angiogenesis; Covalent labeling; Displacement assay; HRP; VEGF.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding, Competitive
  • Biological Assay / methods*
  • Biotin / chemistry
  • Biotin / metabolism
  • Enzyme-Linked Immunosorbent Assay
  • Horseradish Peroxidase / chemistry
  • Horseradish Peroxidase / metabolism*
  • Humans
  • 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*

Substances

  • Ligands
  • Peptide Fragments
  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A
  • Biotin
  • Streptavidin
  • Horseradish Peroxidase
  • FLT1 protein, human
  • Vascular Endothelial Growth Factor Receptor-1