Rabbit anti 6 his tag antibody

The amount of scFv antibody secreted from each yeast strain was evaluated by a western blot analysis. Consistent volumes of BMMY culture supernatants obtained at the final culture point (48 h) for each yeast strain were separated by sodium dodecyl sulphide‐polyacrylamide gel electrophoresis (SDS‐PAGE). Proteins were transferred to a nitrocellulose membrane using a blotting system (iBlot Gel Transfer System, Thermo Fisher Scientific) according to the manufacturer’s instructions, and non‐specific binding was blocked by incubating the membrane in a blocking reagent (BlockingOne, Nacalai Tesque) at room temperature for 1 h. The blocked membrane then was incubated at room temperature for 1 h with rabbit anti‐6‐His Tag antibody (Bethyl Laboratories, Montgomery, TX, USA) diluted 1:5000 in TBS‐T buffer (10 mM Tris‐HCl, pH 8.0, 150 mM NaCl, 0.05% Tween20). After washing the membrane with TBS‐T buffer, scFv was detected by incubation at room temperature for 1 h in alkaline phosphatase (AP)‐conjugated anti‐rabbit IgG (Fc) (Promega, Madison, WI, USA) diluted 1:500 in TBS‐T buffer. After again washing the membrane with TBS‐T buffer, the antibody‐antigen complex was detected by addition of a luminescent substrate (CDP‐STAR Detection Reagent, GE Healthcare, Little Chalfont, UK) and visualized using a charge‐coupled device (CCD) imager (LAS‐4000, GE Healthcare).

The amounts of scFv antibody secreted by each yeast strain were evaluated by western blot analysis. Equal volumes of supernatants from BMMY cultures at the final culture point (48 h) for each yeast strain were separated by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane using a blotting system (iBlot Gel Transfer System, Thermo Fisher Scientific) according to the manufacturer’s instruction, and non-specific binding was blocked by incubation of the membrane in a blocking reagent (BlockingOne, Nacalai Tesque) at room temperature for 1 h. The blocked membrane was then incubated with rabbit anti-6-His tag antibody (Bethyl Laboratories, Inc., Montgomery, TX, USA) in a TBS-T buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.05% Tween20) at a dilution of 1:5000, at room temperature for 1 h. After washing the membrane with TBS-T buffer, anti-rabbit IgG conjugated with an alkaline phosphatase (Promega) was used for scFv detection, at a dilution of 1:5000. After washing with TBS-T buffer, the antibody-antigen complex was visualized on a CCD imager (LAS-4000, GE Healthcare, Little Chalfont, UK) following addition of a luminescent substrate (CDP-STAR Detection Reagent, GE Healthcare).