Anti polyhistidine alkaline phosphatase antibody

A total of 400 µg of each protein was resolved on a 4–12% gradient, 1.5 mm, 15-well Bis-Tris NuPAGE gel in 1X MES buffer (Invitrogen, ThermoFisher). SeeBlue Plus2 pre-stained protein standard (ThermoFisher) was included on the SDS-PAGE for molecular weight reference. Proteins were transferred onto nitrocellulose membranes using the iBlot Western blotting system (Invitrogen, ThermoFisher). Membranes were blocked overnight at 4 °C with 50 mL of 5% non-fat dry milk (Blotting-grade blocker, Bio-Rad Laboratories, Hercules, CA, USA) in 1X tris-buffered saline containing 0.1% Tween-20 (1X TBST). Membranes were probed with either anti-FLAG-alkaline phosphatase antibody (Sigma, A9469, St. Louis, MO, USA) or anti-polyhistidine-alkaline phosphatase antibody (Sigma-Aldrich, catalog #A5588) diluted 1:1000 in 5% non-fat dry milk in 1X TBST for 1 h at 4 °C. Membranes were washed 3 times for 5 min each at room temperature with 1X TBST. Membranes were developed using 1-step NBT/BCIP (ThermoFisher) according to the manufacturer’s recommendations.