Biotin heparan sulfate eu cryptate

42 μL of HS trisaccharide solution in Milli-Q water (0.0038–500 μM) or just Milli-Q water (as a control), and 42 μL of heparanase (5.3 nM, R&D Systems) solution in pH 7.5 triz buffer (consisting of 20 mM TrisHCl, 0.15 M NaCl and 0.1% CHAPS) or just buffer as blank were added into microtubes and pre-incubated at 37 °C for 10 min bringing the [heparanase] to 0.5 nM. Next, 84 μL of biotin-heparan sulfate-Eu cryptate (Cisbio, Cat #: 61BHSKAA) (58.6 ng in pH 5.5 0.2 M NaOAc buffer) was added to the microtubes, and the resulting mixture was incubated for 60 min at 37 °C. The reaction mixture was stopped by adding 168 μL of Streptavidin-XLent! (Cisbio, Cat #: 611SAXLA) (1.0 μg/ml) solution in pH 7.5 dilution buffer made of 0.1 M NaH₂PO₄, 0.8 M KF, 0.1% BSA. After the mixture had been stirring at room temperature for 15 min, 100 μL (per well) of the reaction mixture was transferred to a 96 well microplate (Corning #3693 96 well, white polystyrene, half-area) in triplicates and HTRF emissions at 616 nm and 665 nm were measured by exciting at 340 nm using a SpectraMax iD5 Microplate Reader (Molecular Devices).

A total of 42 μL of inhibitor solution in Milli-Q water (0.00016–4000 μM) or just Milli-Q water (as a control) and 42 μL of heparanase (5.3 nM, R&D Systems) solution in pH 7.5 tris buffer (consisting of 20 mM Tris HCl, 0.15 M NaCl, and 0.1% CHAPS) or just buffer as a blank were added into microtubes and preincubated at 37 °C for 10 min. Next, 84 μL of biotin-heparan sulfate-Eu cryptate (Cisbio, Cat # 61BHSKAA; 58.6 ng in pH 5.5 0.2 M NaCH₃CO₂ buffer) was added to the microtubes, and the resulting mixture was incubated for 60 min at 37 °C. The reaction mixture was stopped by adding 168 μL of Streptavidin-XLent! (Cisbio, Cat # 611SAXLA; 1.0 μg/mL) solution in pH 7.5 dilution buffer made of 0.1 M NaPO₄, 0.8 M KF, and 0.1% BSA. After the mixture had been incubating at room temperature for 15 min, 100 μL (per well) of the reaction mixture was transferred to a 96-well microplate (Corning #3693 96-well, white polystyrene, half area) in triplicates and HTRF emissions at 616 and 665 nm were measured by exciting at 340 nm using SpectraMax i3x Microplate Reader (Molecular Devices).