Anti his tag mab hrp direct

After SDS-PAGE, proteins were electroblotted onto a Hybond-ECL membrane (GE Healthcare, Buckinghamshire, UK), which was blocked in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and 5% skimmed milk. Proteins on the membrane were probed with antibody in blocking buffer and then the membrane was washed with TBS-T. Rabbit polyclonal anti-AcK antibody (ImmuneChem, Burnaby, BC, Canada) and Anti-His-tag mAb-HRP-DirecT (Medical & Biological Laboratories, Aichi, Japan) were used at a 1/3000 and 1/10,000 dilution respectively as primary antibodies. Anti-Rabbit IgG HRP conjugate (Promega, Madison, WI, USA) was used as the secondary antibody. Proteins on the membrane were probed with the primary antibody in blocking buffer and then washed with TBS-T. Proteins were detected by Immobilon Western Chemilum HRP substrate (Merck Millipore) and visualized on a LAS 4000 (GE Healthcare).

For the expression of XeHypBA1, vector pET23-xcv2724 was transferred into E. coli BL21 (λDE3) cells, then a single colony was added to 50 mL LB containing 50 mg/mL kanamycin and grown to an OD₆₀₀ of 0.6 at 37°C. The culture was then induced with 1 mM isopropyl ß-d-1-thiogalactopyranoside (IPTG) for 18 h at 15°C. For the expression of XeHypBA2, vector pCold-xcv2729 was transferred into E. coli BL21 (λDE3) cells, and the protein fused with the trigger factor chaperone was expressed as described above. For the expression of XeHypAA, vector pBIC3-xcv2728 was transferred into B. choshinensis SP3, and a single colony was grown in 50 mL TM containing 50 mg/mL neomycin at 30°C for 48 h.
Expressed proteins were purified with the MagneHis Protein Purification System (Promega, Madison, WI) or the Capturem His-Tagged Purification kit (Takara Bio) and desalted with the Zeba Spin Desalting Columns 7K MWCO (Thermo Scientific, Rockford, IL, USA). The purified proteins were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or Western blotting with Anti-His-tag mAb-HRP-DirecT (Medical & Biological Laboratories, Nagoya, Japan).