5 bromo 4 chloro 3 indolyl phosphate bcip

The rSUB, rFER2, and rP0 proteins were verified by SDS-PAGE in a 16% gel under a reduced condition and quantified using a Nano-Drop spectrometer (Nano-Q; OPTIZEN). For Western blot [45 (link)], 5 µg of protein sample was mixed with 15 µl loading buffer (5% SDS, 5% Tris [pH 6.8], 0.2% bromophenol blue and 20% glycerol) and heated at 100 °C for 10 min. Each protein sample (20 µl), protein marker (5 µl; Bio-Rad Laboratories) and BSA standard (5 µl) were gel-electrophoresed at 100 V for 20 min in stacking gel and 150 V for 1 h in running gel, at 4 °C. The protein samples were then transferred to a nitrocellulose membrane in 12 mM carbonate buffer (pH 9.9) at 70 V for 1 h at 4 °C. The membranes were blocked with blocking buffer (1× PBS containing 5% skim milk) and incubated with shaking at room temperature (RT) for 1 h, followed by 3 washes with the same buffer. The membranes were then treated with 1:1000 dilution of rabbit-anti-His monoclonal antibodies (GenScript). Detection was performed in alkaline phosphatase buffer (100 mM Tris–HCl, 100 mM NaCl, 5 mM MgCl2) with the addition of 0.3 mg/ml nitro blue tetrazolium (NBT; Thermo Fisher Scientific) and 0.15 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Thermo Fisher Scientific) [16 (link)].

Cell lysates were boiled in 1× Laemmli sample buffer for 5 min, and then electrophoresed on 4–20% Tris-Glycine eXtended (TGX) precast protein gels (Biorad). Proteins were transferred to 0.2 μm polyvinylidene difluoride (PVDF) membranes by using a Trans-Blot Turbo Transfer System (Biorad). The membranes were blocked for 30 min in 5% non-fat milk, and then incubated for 2 hours at room temperature or overnight at 4 °C with a primary antibody. After 3 times of 5-min wash with TBST, the membranes were immunoblotted with alkaline phosphatase (AP) conjugated goat-anti-rabbit IgG or goat-anti-mouse IgG secondary antibody (Promega) for 1 hour at room temperature. After 3 times of 5-min wash with TBST, blots were developed using 5-Bromo-4-chloro-3-indolyl phosphate (BCIP, ThermoFisher) and Nitro blue tetrazolium (NBT, ThermoFisher) as substrates. The blots were then imaged on a ChemiDoc Imaging System (Biorad) and the bands on blots were quantified by ImageJ (Open source Java program from NIH) or the immunoblotting quantification software Image Studio™ Lite (LI-COR Biosciences).