Amersham protran premium 0.45 μm nitrocellulose

Total protein in urine and stool samples was quantified using a Pierce BCA protein assay (cat. no. 23277) in triplicate according to manufacturer’s protocol. Forty micrograms of total protein from urine and stool samples and 10 μL of plant sap extract supernatant were separated on 4–12% SDS-PAGE precast gels in 1× MOPS buffer (Thermo Fisher Scientific) for 40 min at 200 V and 120 mA. SeeBlue Plus2 (Thermo Fisher Scientific) was used as a marker. Proteins were then electrotransferred onto a nitrocellulose membrane (Amersham Protran Premium 0.45 μm nitrocellulose, GE Healthcare). The membranes were blocked with 5% (w/v) BSA in PBS (room temperature, 1 h) and then washed with PBS. Primary antibody incubation proceeded with 1:1000 dilution of rabbit anti-CPMV polyclonal antibody (PAC 12273/12274) in 5% (w/v) BSA in PBS with 0.05% (v/v) Tween-20 (PBS-T buffer at room temperature, 1 h) followed by washing three times with PBS-T. Secondary antibody incubation proceeded with HRP-labeled goat anti-rabbit IgG (H+L) (Fisher Scientific) in 2% (w/v) BSA in 1× PBS-T (room temperature, 1 h) followed by washing three times with PBS-T and one time with PBS. Membranes were developed using Thermo Scientific Pierce ECL Western Blotting substrate for 1 min and imaged using chemiluminescence on ProteinSimple FluorChem R.