Heparin biotin

BLI assays were performed on an Octet Red Instrument (fortéBIO) at 25 °C. Immobilization and binding analysis were carried out at 1000 rpm using HBS-EP buffer [10 mM HEPES, pH 7.4, 150 mM NaCl, 3.0 mM EDTA, and 0.005% (v/v) surfactant tween20]. A solution affinity assay, used to determine affinities of ligands by SPR analysis was adopted to BLI.³⁹ In this method, protein is mixed with various concentrations of ligand (glycopolymer 20 or heparin, 18 kDa). Free protein in this equilibrium mixture is tested for binding against immobilized heparin (all proteins are carrier-free and purchased from R&D Systems). Heparin-biotin (Creative PEGworks, 18 kDa, 1 biotin per HP polymer), 5 μg /mL was immobilized on to streptavidin biosensors (fortéBio) for 5 min. Binding experiments were carried under conditions of mass transport. Binding was fitted to equation 1 using Graphpad Prism.^{40 (link)} BLI response was utilized in place of F and ligand (heparin/glycopolymer 20) concentration was used in place of [metal]. Binding analysis of P-selectin was carried out with HBS-EP buffer with 2 mM CaCl₂, 2 mM MgCl₂ and 0.5mg/ mL BSA.

All commercial chemical reagents used for synthesis were used as received from Sigma Aldrich, Alfa Aesar, TCI, and Combi-Blocks, unless otherwise mentioned. Other reagents and materials were purchased from the following: heparanase, FGF-1, FGF-2, P-selectin, and ATIII were all carrier-free (R&D Systems), HUVECs and their reagents (Lonza), Heparin-biotin (Creative PEGworks), Streptavidin BLI biosensors (fortéBIO), CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Fisher Scientific), TR-FRET heparanase inhibition kit (Cis-bio).

Assays were performed on an Octet Red Instrument (fortéBIO) at 25 °C. Immobilization and binding analysis were carried out at 1000 rpm using HBS-EP buffer [10 mM HEPES, pH 7.4, 150 mM NaCl, 3.0 mM EDTA, and 0.005% (v/v) surfactant tween20]. A solution affinity assay, used to determine affinities of ligands by SPR analysis was adopted to BLI.⁴⁶ In this method, PF 4 (100 nM; 100 μL) was mixed with various concentrations of trisaccharide 1α (0.08‐250 μM; 100 μL) or heparin (0.4‐400 nM; 100 μL). Free protein in this equilibrium mixture is tested for binding against immobilized heparin (all proteins are carrier-free and purchased from R&D Systems). Heparin-biotin (Creative PEGworks, 18 kDa, 1 biotin per HP polymer), 5 μg /mL was immobilized on to streptavidin biosensors (fortéBio) for 5 min. Binding experiments were carried under conditions of mass transport. Binding was fitted to equation 1 of ref 10 (link) using Graphpad Prism. BLI response was used in place of F and ligand (heparin /trisaccharide 1α) concentration was used in place of [metal].