Substrate for enhanced chemiluminescence

Analysis of the particle distribution after ultracentrifugation was carried out by SDS-PAGE using 4-to-12% gradient Bis-Tris gels in morpholineethanesulfonic acid (MES)-SDS running buffer. After electrophoresis, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane by electroblotting, and subsequently, the membranes were blocked overnight at 4°C with 3% nonfat milk in TBST (Tris-buffered saline, 0.1% [vol/vol] Tween 20). After blocking, the membranes were incubated for 1 h at RT with anti-sHBsAg pAb (OriGene) or mAb AP33 diluted in 0.3% nonfat milk in TBST, washed with TBST, and then incubated with secondary horseradish peroxidase (HRP)-conjugated antibodies (Santa Cruz Biotechnology). The results were developed using substrate for enhanced chemiluminescence (Thermo Scientific).

An analysis of the particle expression was carried out by SDS-PAGE of Leishmania tarentolae cell lysates using 4–12% gradient Bis–Tris gels in MES SDS running buffer. After electrophoresis, proteins were transferred onto a PVDF membrane by electroblotting, and subsequently the membranes were blocked overnight at 4 °C with 3% nonfat milk in TBST [TBS buffer, 0.1% (v/v) Tween-20]. Following blocking, the membranes were incubated for 1 h at room temperature (RT) with primary anti-HBsAg antibodies diluted in 3% nonfat milk in TBST, washed with TBST, and then incubated with goat anti-rabbit secondary horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology, Dallas, TX, USA). The results were obtained through development in the substrate for enhanced chemiluminescence (Thermo Scientific).

The E1E2 heterodimers with or without N417 expressed in HEK293 cells were divided into two equal groups: one for digestion with PNGase F and one for the undigested control. The digestions were conducted for 16 h under native conditions at 37°C in the buffer provided by the manufacturer (New England BioLabs). The digested samples and the controls were separated by SDS-PAGE as described above and analyzed by Western blotting using mouse sera (diluted 1:1,000), AP33 (diluted 1:2,500), and ALP98 (diluted 1:1,500). Goat anti-mouse secondary HRP-conjugated antibodies (Santa Cruz Biotechnology) were used for detection. The results were developed using substrate for enhanced chemiluminescence (Thermo Scientific).