Anti histidine

For Western blot analysis, purified protein was incubated at 100°C for 5 min in loading buffer, separated by SDS-PAGE (12% gel) and transferred onto PVDF membrane (GE) using an Electro-transfer unit (Bio-Rad). The membranes were sequentially incubated with mouse polyclonal anti-rTgPI-1 antibody (1:1000) or anti-Histidine (1:3000; Sigma) and alkaline phosphatase conjugated goat anti-mouse IgG (1:5000; Sigma). After washing, the reaction was developed by the addition of nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) substrate. PageRuler^TM Prestained Protein Ladder (Thermo Scientific) was used as molecular marker.

Equal amounts of protein were run on 15% SDS-PAGE, transferred to PVDF (BDH), and immunodetected by anti-histidine (Sigma-Aldrich) primary antibody followed by peroxidase-coupled secondary antibody (Sigma-Aldrich). Proteins were visualized by enhanced chemiluminescence (Chemidoc, Bio-Rad). For kinetic constant determination, SP-1 (20 nM) was incubated at room temperature, in a microtiter plate, with assay buffer (20 mM Tris, 300 mM NaCl, 1 mM CaCl₂, pH 8) and varying concentrations (0.05–2 mM) of synthetic substrates in a final volume of 100 μL. Activity measurements were performed using a plate reader (Synergy™ 2 Multi-Mode Microplate Reader, BioTek^®) set at (410 nm), for released ANB-NH₂, or (Excitation: 360 nm, Emission: 460 nm), for the fluorogenic substrates. Enzyme-free reactions were used as negative controls. All experiments were performed in triplicate. Standard curves were used to calculate the rate of product generation. Data were processed by non-linear regression analysis with GraphPad Prism 8.0, GraphPad Software (San Diego, CA, USA) and globally fitted to the Michaelis–Menten equation.