Ultra tmb blotting solution

Western blot analysis was performed as previously described⁴⁷ . In brief, crude protein was extracted and isolated before mixing with 6X protein loading dye and boiling for 10 min. The stained protein was run under 12.5% SDS-PAGE and transferred to a Hybond PVDF membrane (GE Healthcare) in a semi-dry transfer cell (Bio-Rad). The transferred membrane was blocked and hybridized with anti-6His-peroxidase primary antibody (Roche, Switzerland) and developed with Ultra TMB-Blotting Solution (Thermo Scientific) according to the manufacturer’s recommendation.

All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University (MUTM 2020–058-02). Informed consent was obtained from all subjects and/or their legal guardian(s). Proteins from SDS-PAGE were transferred onto a nitrocellulose membrane. The membrane was cut into five strips and immunoblotting was performed using sera from five different patients as the primary antibody. The proteins separated by 2-DE were transferred onto a nitrocellulose membrane and pooled patient sera was used as the primary antibody. Membranes were blocked using 5% (w/v) non-fat milk in phosphate buffered saline (PBS) for 2 h at room temperature, then rinsed with PBS containing 0.05% (v/v) Tween-20. Serum samples diluted 1:200 in PBS containing 1% non-fat milk were added to the membranes and incubated overnight at 4 °C. After incubation, the membranes were washed three times with PBS containing 0.05% (v/v) Tween-200. Then horseradish peroxidase-conjugated goat anti-human IgG secondary antibodies were added and incubated for 1 h. Immunogen spots were visualized by detection of peroxidase activity using the Ultra TMB-Blotting Solution (ThermoFisher Scientific, UK). Immunoreactive protein spots were excised from silver-stained 2-DE gels and subjected to in-gel digestion.

The separated polypeptide spots from 2DE gels were transferred to nitrocellulose membrane for 90 min at 18 V on a Trans-blot semi-dry Transfer CellTM (Biorad) in semi-dry transfer buffer (48 mM Tris and 2.93 g glycine) pH 9.2 containing 20% methanol. The membranes were blocked using 5% (w/v) non-fat milk in PBS for 2 h at room temperature. The membranes were rinsed twice with PBS-T buffer pH 7.4 (8 mM sodium phosphate, 2 mM potassium phosphate, 140 mM NaCl, 2.7 mM KCl and 0.5% v/v Tween) for 30 s each. The blotted membranes were incubated with either monovalent or polyvalent antivenom (1:1000 in 0.2% BSA-PBS). After washing the membrane three times with PBS-T, 50 μL of horseradish peroxidase-conjugated goat anti-horse-IgG (Abcam, Cambridge, UK) in PBS-T (1:2000) was added, and the mixture was incubated for 1 h at ambient temperature under constant agitation. Membranes were washed three times with PBS-T buffer and one time with PBS. Immunogen spots were visualized by detection of peroxidase activity using Ultra TMB-Blotting Solution (ThermoFisher Scientific, UK).