Streptavidin dynabeads m 280 streptavidin

SdAb binding to soluble hemin was assessed by a pull-down assay. Briefly, sdAbs were incubated (5 h, 4 °C, agitation) with heme-biotin and half of the reaction mixture was incubated (2 h, RT, agitation) with of Streptavidin-Dynabeads (M-280 Streptavidin; Invitrogen by Thermo Fischer Scientific), previously blocked (5 h, RT) with protein-free blocking buffer (Pierce). Mixture was washed (3×; 5 min; PBS, 0.05% Tween 20) and magnetic beads were captured (5 min magnetic field) according to the manufacture's instructions. SdAbs-heme-biotin complexes were collected (1 min, magnetic field) and incubated (20 min, 100 °C, agitation) in loading buffer (50 mm Tris-HCl pH 6.8, 2% SDS, 10% Glycerol, 1% β-ME, 12.5 mm EDTA, 0.02% Bromophenol blue) (pull-down). The other half of the reaction mixture was denatured (100 °C, 10 min) in loading buffer. Proteins were applied into a 15% SDS/PAGE gel under denaturing conditions and stained with a Coomassie-based stain (Instant Blue, Gentaur, 30 min, RT). Alternatively, sdAbs were detected by Western blotting using a rat anti-HA HRP conjugated mAb (1/5000; Roche^®) (1 h, RT).

SdAb 2H10 was incubated (30–60 min, 4 °C, agitation) with heme-biotin and HPX was added to the mixture at 1/6 SdAb/HPX molar ratio (30–60 min, 4 °C, agitation). To evaluate the ability of HPX to bind heme-biotin, HPX was incubated (30–60 min, 4 °C, agitation) with heme-biotin. Half of the reaction mixture was incubated (2 h, RT, agitation) with Streptavidin-Dynabeads (M-280 Streptavidin; Invitrogen), previously blocked (5 h, RT) with protein-free blocking buffer (Pierce). The reaction mixture was washed (3 ×; 5 min; PBS, 0.05% Tween 20), magnetic beads were captured (5 min magnetic field) according to the manufacture's instructions and incubated (20 min, 100 °C, agitation) in loading buffer (50 mm Tris-HCl pH 6.8, 2% SDS, 10% Glycerol, 1% β-ME, 12.5 mm EDTA, 0.02% Bromophenol blue). The other half of the reaction mixture was denatured (100 °C, 10 min) in loading buffer. Reaction mixtures in loading buffer were applied into a 15% SDS/PAGE gel under denaturing conditions and stained with a coomassie-based stain (Instant Blue, Gentaur, 30 min, RT).