Semi dry blotting system

Recombinant proteins were analyzed by 13% SDS-PAGE under reducing conditions. The separated proteins were transferred onto a 0.45-μm PVDF membrane (Millipore) in semidry transfer buffer (50 mM Tris, 190 mM glycine, 3.5 mM SDS, 20% methanol) at a constant 400 mA for 40 min using a semidry blotting system (ATTO Corp., Tokyo, Japan). The PVDF membrane (Cytiva, Marlborough, MA) containing recombinant protein was blocked with 5% skim milk in PBS-T (0.5% v/v Tween-20 in 1× PBS) and then incubated with anti-GST antibody (Novagen, Reno, NV) and mouse and rabbit immune sera diluted 1:2,000 in PBS-T. After the primary antibody reaction, the membrane was incubated with the secondary IRDye^® goat anti-mouse (1: 5,000 dilution) or IRDye^® goat anti-rabbit (1: 5,000) (Li-COR^® Bioscience, Lincoln, NE) antibodies to detect antigens. The results were visualized in the Odyssey infrared imaging system and analyzed with Odyssey software (Li-COR^® Bioscience) (Lee et al., 2016 (link)).

The parasite proteins were extracted in reducing sample buffer for SDS-PAGE. Five micrograms of recombinant PvRALP1-Ecto or PvRALP1-Tr protein were loaded into each well and separated by SDS-PAGE under reducing conditions. The separated proteins were transferred to 0.45 μm polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) in a semidry transfer buffer (50 mM Tris, 190 mM glycine, 3.5 mM SDS, 20% methanol) at 400 mA for 40 min using a semidry blotting system (ATTO Corp., Tokyo, Japan). After blocking with 5% skim milk in phosphate-buffered saline containing 0.2% Tween 20 (PBS-T), the membranes were probed with mouse anti-PvRALP1-Ecto and anti-PvRALP1-Tr sera, rabbit anti-PvRALP1-Tr serum, anti-GST monoclonal antibody (Novagen, Madison, WI, USA), anti-penta-His monoclonal antibody (Qiagen), preimmune mouse serum, pooled sera from P. vivax malaria patients or noninfected individuals, all diluted 1:200 in PBS-T. IRDye goat anti-mouse, IRDye goat anti-rabbit, or IRDye goat anti-human sera (LI-COR Biosciences, Lincoln, NE, USA) were used to detect recombinant proteins according to the manufacturer’s instructions. Data were scanned with an Odyssey infrared imaging system (LI-COR Biosciences) and analysed with Odyssey software (LI-COR Biosciences).

The recombinant PvMSA180 proteins were resolved on 13% SDS-PAGE gels under reducing conditions, and then electrotransferred to 0.45 µm PVDF membranes (Millipore, Billerica, MA, USA) in semi-dry transfer buffer (50 mM Tris, 190 mM glycine, 3.5 mM SDS, 20% methanol) at a constant current of 360 mA for 40 min using a semi-dry blotting system (ATTO Corp., Tokyo, Japan). Recombinant PvMSA180-D1 and PvMSA180-D4 (1 µg each) and PvDBP-RII (0.5 µg) were used to assay antibody responses. The membranes were blocked in 5% skim milk and then incubated with a primary anti-GST antibody (1:10,000), anti-pentahistidine antibody (1:2000), mouse immune serum, or pooled patient serum (1:100), followed by incubation with a secondary IRDye^® goat anti-mouse (1:10,000 dilution) or IRDye^® goat anti-human (1:20,000) (LI-COR^® Bioscience, Lincoln, NE, USA) antibody. An Odyssey infrared imaging system and the accompanying software (LI-COR^® Bioscience) were used for data analysis.