Heparin biotin sodium salt

Wild-type HSA-DKK2C2 (ACE464) and each of the HS-binding variants were examined for binding to heparin–biotin using ELISA. Nunc clear flat-bottom immuno non-sterile 96-well plates (ThermoFisher Scientific) were coated with 15 µg/ml of each of the HSA-DKK2C2 variants and incubated overnight at 4°C. Following three washes with PBST (20 mM Na₂HPO₄, 150 mM NaCl, 0.05% Tween-20), wells were incubated with ELISA blocking buffer HBSS (25 mM HEPES pH 7.0, 1% bovine serum albumin (BSA), 0.1% ovalbumin, 0.1% non-fat dry milk (NFDM), 0.001% NaN₃) at RT for 1 h. Following three washes with PBST, wells were incubated at RT for 1 h with heparin–biotin sodium salt (Sigma-Aldrich) in a concentration series starting at 50 µg/ml (~4 µM), preceded by eight 5-fold dilutions in PBST, 0.05% BSA. Following three washes with PBST, wells were incubated at RT for 10 min with streptavidin-HRP (ThermoFisher Scientific) in a 1:8000 dilution in PBST, 0.05% BSA. Following two washes with PBST, wells were incubated at RT for 20 min with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (0.1 M sodium acetate citric acid pH 4.9, 0.42 mM TMB, 0.004% H₂O₂). Developed ELISAs were stopped by the addition of 2 N H₂SO₄ and plates were scanned at 450 nm using a Molecular Devices SpectraMax M5 microplate reader. IC50 values were calculated with Softmax Pro v5.4.4 software.