Hrp conjugated anti his tag antibody

To verify the binding ability and its structure specificity of β-1,6-glucanase variants to glucans, direct and competitive ELISA-like assays were carried out. In brief, for direct ELISA, pustulan or laminarin (0–5,000 ng/ml) in 0.1 m sodium carbonate buffer (pH 9.5) was added to a 96-well clear plate and incubated at 4 °C. The next day, the plate was washed by PBS containing 0.05% Tween 20 (PBST) and blocked with 1% BSA/PBST (BPBST) by incubating for 1 h. Solid-phased glucans were reacted with recombinant modified Neg1 in BPBST (2 μg/ml) for 1 h and washed, and the HRP-conjugated anti-His tag antibody (BioLegend) was added to the plate. After 1 h, the plate was washed, and the binding of modified enzymes to solid-phase glucans was monitored using the peroxidase substrate TMB, and color development was stopped with 1 m phosphoric acid; the optical density was measured at 450 nm using a microplate reader (MTP450; Corona Electric, Ibaraki, Japan). For competitive ELISA, various glucans (20 and 100 μg/ml, final concentrations) were mixed with Neg1–E321Q–His (0.5 μg/ml, final concentration) and preincubated for 1 h. The pustulan (0.5 μg/ml)-coated 96-well clear plate was blocked, washed, and incubated with the above mixture of E321Q–His and glucan for 1 h. After washing, the binding of E321Q–His to the immobilized pustulan was assessed as described above.