TSP were separated by 10% SDS-PAGE, transferred to a PVDF membrane, and then incubated with mouse anti-c-Myc monoclonal antibody (Genscript, A00864, Piscataway, USA). ONE-HOUR Western Basic Kit Mouse (GeneScript, Piscataway, EUA) and Clarity Western ECL Substrate (Bio-Rad, Mississauga, Canada) were used to detect the recombinant proteins in a MicroChemi 4.2 (DNR Bio-Imaging Systems, Jerusalem, Israel). Western blots were analyzed using image densitometry with TotalLab TL100 software (Nonlinear Dynamics, Durhan, USA). The NS1 constructs were quantified by comparison with known amounts of a synthetic positive control protein containing a cellulose-binding domain (CBD) and a c-Myc tag (synthesized by Genscript, Piscataway, USA).
The molecular identity of the produced protein was further validated by western blotting analysis under non-reducing conditions against a specific antibody. Therefore, NS1 ELP-ER protein was purified by affinity chromatography using the C-Myc Tagged Protein Mild Purification Kit (MBL, 3305, Woburn, USA) according to the manufacturer's instructions. A monoclonal antibody against the full length native DENV NS1 glycoprotein (Abcam, ab41616) was used for protein detection (1:100 dilution).
The molecular identity of the produced protein was further validated by western blotting analysis under non-reducing conditions against a specific antibody. Therefore, NS1 ELP-ER protein was purified by affinity chromatography using the C-Myc Tagged Protein Mild Purification Kit (MBL, 3305, Woburn, USA) according to the manufacturer's instructions. A monoclonal antibody against the full length native DENV NS1 glycoprotein (Abcam, ab41616) was used for protein detection (1:100 dilution).