A fluorescence polarization assay for identifying ligands that bind to vascular endothelial growth factor

Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
Analytical Biochemistry (Impact Factor: 2.22). 08/2008; 378(1):8-14. DOI: 10.1016/j.ab.2008.03.043
Source: PubMed

ABSTRACT Vascular endothelial growth factor (VEGF) is a homodimeric proangiogenic protein that induces endothelial cell migration and proliferation primarily through interactions with its major receptors, VEGFR-1 and VEGFR-2. Inhibitors of one or both of these VEGF-receptor interactions could be beneficial as therapeutics for diseases caused by dysfunctional angiogenesis (e.g., cancer). Others have reported small peptides that bind to the VEGF dimer at surface regions that are recognized by the receptors. Here we report the development of a fluorescence polarization assay based on the binding to VEGF of a derivative of one of these peptides that has been labeled with BODIPY-tetramethylrhodamine (BODIPY(TMR)). This 384-well format assay is tolerant to dimethyl sulfoxide (DMSO, up to 4% [v/v]) and has a Z' factor of 0.76, making it useful for identifying molecules that associate with the receptor-binding surface of the VEGF dimer.

Download full-text


Available from: Richard C Willson, Sep 27, 2015
29 Reads
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Early reports on the identification of fluorescent probes for receptors date back to mid-1970s. Fluorescent probes were initially used to visualize molecular targets in an analogous way to the use of fluorescent antibodies but with the same resolution as isotopically labelled ligands. In parallel to the rapid development of techniques, such as fluorescence correlation spectroscopy, multi-photon excitation fluorescence microscopy, fluorescence polarization and in vivo fluorescence imaging, fluorescent probes are becoming multifaceted tools in life science. The present review will focus on how the design of fluorescent ligands for receptors has evolved to meet the needs of most recent fluorescence applications.
    Drug discovery today 08/2009; 14(13-14):706-12. DOI:10.1016/j.drudis.2009.03.015 · 6.69 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The history of the fluorescence polarization/anisotropy in diagnostics and imaging and discuss in detail both theoretical and practical aspects has been reported. Light can be considered as oscillations of an electromagnetic fields characterized by electric and magnetic components perpendicular to the direction of light propagation. The utility of fluorescence polarization in the clinical and biomedical sciences ultimately rests on the dependence of the observed polarization on the rotational diffusion rate of molecules. The most fundamental source of error in fluorescence polarization/anisotropy determinations would be actual errors in the polarization measurements themselves. Fluorescence polarization/ anisotropy offers significant advantages for some applications in clinical chemistry and bioassays and its use in biotechnology and drug discovery will no doubt continue to expand.
    Chemical Reviews 03/2010; 110(5):2685-708. DOI:10.1021/cr900267p · 46.57 Impact Factor
Show more