A fluorescence polarization assay for identifying ligands that bind to vascular endothelial growth factor
Section snippets
General
Fmoc-l-α-amino acids, O-benotriazole-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), and NovaSyn TGR resin were purchased from NovaBiochem (San Diego, CA, USA). 6-Carboxyfluorescein was purchased from Anaspec (San Jose, CA, USA). BODIPYTMR-SE was purchased from Invitrogen (Carlsbad, CA, USA). (2-[2-(Fmoc-amino)-ethoxy]-ethoxy)-acetic acid (Fmoc-AEEAc-OH) was purchased from Bachem (Torrance, CA, USA). Piperidine, 1-hydroxybenzotriazole (HOBt), trifluoroacetic acid (TFA),
VEGF construct selection and validation
We considered several isoforms of VEGF-A as we were designing the FP assay. High-resolution structures of VEGF8–109 alone [39] and in complex with a soluble fragment of VEGFR-1 [17] or with the peptide v107 [49] have been solved by researchers at Genentech. In addition, VEGF8–109 was used with phage-displayed libraries to identify peptide ligands v107 and v114 [32]. However, VEGF165 (residues 1–165) is the most prevalent isoform in vivo [2]. This protein includes a heparin-binding domain,
Conclusion
We have developed an FP assay that can be used to screen for molecules that bind to VEGF165 at the VEGF receptor-binding site, which is shared by small peptides v107 and v114. This is the first demonstration of a fluorescence-based assay for VEGF that is suitable for use in a high-throughput format. This assay has several advantages over other assay modes such as having all components free in solution and not relying on radioactive materials for quantification. Blocking the interaction between
Acknowledgments
This research was supported by the National Institutes of Health (NIH) (GM56414 to S.H.G., DK50107 to E.H.B., and EY16995 to N.S.). R.C.W. was supported by the NIH and the Welch Foundation. We thank W. Seth Horne and Melissa D. Boersma for assistance with FP assay design and helpful discussions. The plate reader used for FP assays is in the University of Wisconsin–Madison W. M. Keck Center for Chemical Genomics. K.J.P. was supported in part by the NIH Chemistry–Biology Interface Training
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