Skip to Content
MilliporeSigma
  • Real-Time Ligand Binding of Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in Living Cells.

Real-Time Ligand Binding of Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in Living Cells.

Cell chemical biology (2018-07-31)
Chloe J Peach, Laura E Kilpatrick, Rachel Friedman-Ohana, Kris Zimmerman, Matthew B Robers, Keith V Wood, Jeanette Woolard, Stephen J Hill
ABSTRACT

Fluorescent VEGF-A isoforms have been evaluated for their ability to discriminate between VEGFR2 and NRP1 in real-time ligand binding studies in live cells using BRET. To enable this, we synthesized single-site (N-terminal cysteine) labeled versions of VEGF165a, VEGF165b, and VEGF121a. These were used in combination with N-terminal NanoLuc-tagged VEGFR2 or NRP1 to evaluate the selectivity of VEGF isoforms for these two membrane proteins. All fluorescent VEGF-A isoforms displayed high affinity for VEGFR2. Only VEGF165a-TMR bound to NanoLuc-NRP1 with a similar high affinity (4.4 nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF165a > VEGF189a > VEGF145a. VEGF165b, VEGF-Ax, VEGF121a, and VEGF111a were unable to bind to NRP1. There were marked differences in the kinetic binding profiles of VEGF165a-TMR for NRP1 and VEGFR2. These data emphasize the importance of the kinetic aspects of ligand binding to VEGFR2 and its co-receptors in the dynamics of VEGF signaling.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal Anti-Vascular Endothelial Growth Factor Receptor-1 antibody produced in mouse, clone FLT-19, tissue culture supernatant
Sigma-Aldrich
Chicken Serum, USA origin, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Albumin, Bovine Serum, Fraction V, Fatty Acid-Free, Nuclease- and Protease-Free, BSA Fatty Acid-free is designed for use in serological testing, RIA, and hormone response studies. Suitable for use in Molecular Biology applications, such as Northern and Southern blots.
Roche
Bovine Serum Albumin Fraction V, protease-free, from bovine serum
Sigma-Aldrich
Monoclonal Anti-Vascular Endothelial Growth Factor Receptor-2 antibody produced in mouse, clone KDR-1, ascites fluid
Sigma-Aldrich
Donkey serum