Power blotter semi dry transfer system

The four plasmid constructs were transformed into E. coli XL1-Blue, Origami 2, and Origami (DE3) by incubating competent cells with each plasmid under the general conditions for heat shock treatment. Using harvested cells after the culture of the transformed cells, total and soluble fractions of the expressed proteins were prepared as described in the analysis of the expression pattern of mCherry-fused scvhFGF19 variants. The expression patterns of scvhFGF19 were analyzed using SDS-PAGE and Western blotting. For Western blot analysis, the proteins separated by SDS-PAGE (15%) were transferred into a nitrocellulose membrane (GE healthcare, Chicago, IL, USA) using the Power Blotter-Semi-dry transfer system (ThermoFisher scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The membrane was then blocked using 5% skim milk for 1 h, followed by overnight probing at 4 °C with anti-human FGF19 monoclonal antibody (1:5000, R&D Systems, Minneapolis, MN, USA). Next, the membrane was incubated with horseradish peroxidase-linked goat anti-mouse immunoglobulin G (1:5000, Enzo Life Sciences, Ann Arbor, MI, USA) at room temperature for 1 h. The proteins on the membrane were visualized using the ECL kit detection system (Bio-Rad, Hercules, CA, USA).

Western blot assays were performed with Power Blotter Semidry Transfer System (Thermo Fisher Scientific). To control the correct transfer, nitrocellulose membranes were stained with Pierce Reversible Protein Stain Kit for nitrocellulose membranes (Thermo Fisher Scientific), and the image was acquired with Alliance 9.7 Western Blot Imaging System (UVITEC). Membranes were blocked with 4% skimmed milk (nonfat dry milk) or 3% BSA in PBS and 0.1% or 0.01% Tween-20, depending on the antibody. The blocking step lasted for around 1 h. The primary antibody was incubated overnight at 4 °C and, the day after, three washes with PBS and 0.1% or 0.01% Tween-20 were performed (5 min each).