NOMV preparations were analysed by HPLC-SEC with a TosoHaas TSK gel 6,000 + 4,000 PW columns equilibrated in PBS, and with in-line UV, fluorescence emission, and MALS (Dawn Heleos II, Wyatt) detectors. 80 μL of samples, containing 0.1 mg/mL total protein, were injected and eluted with a flow rate of 0.5 mL/min in PBS. UV, fluorescence emission, and multiangle light scattering (MALS) data were collected and analysed using Empower 3 and ASTRA 6 software (Wyatt Technology). Light scattering analysis was done using “Zimm” model with fit degree 1.
Dynamic light scattering (dls) measurements were performed with a Malvern Zetasizer Nano ZS (Malvern, Germany), equipped with a 633-nm He-Ne laser and operating at an angle of 173°. The software used for collecting and analysing data was the Setasizer 7.11 from Malvern. 40 μL of each sample were measured in ZEN 0040-disposable micro cuvette (Malvern). The measurements were done with an automatic attenuator, and at controlled temperature of 25°C. For each sample, 10–15 runs of 10 s were performed, with 3 repetitions. The intensity size distribution, the Z-average diameter, and the polydispersity index (PdI) were obtained from the autocorrelation function using the “general purpose mode”. Default lower threshold of 0.05 and upper threshold of 0.01 were used. Viscosity and refractive index of PBS at 25°C were used for data analysis.
Dynamic light scattering (dls) measurements were performed with a Malvern Zetasizer Nano ZS (Malvern, Germany), equipped with a 633-nm He-Ne laser and operating at an angle of 173°. The software used for collecting and analysing data was the Setasizer 7.11 from Malvern. 40 μL of each sample were measured in ZEN 0040-disposable micro cuvette (Malvern). The measurements were done with an automatic attenuator, and at controlled temperature of 25°C. For each sample, 10–15 runs of 10 s were performed, with 3 repetitions. The intensity size distribution, the Z-average diameter, and the polydispersity index (PdI) were obtained from the autocorrelation function using the “general purpose mode”. Default lower threshold of 0.05 and upper threshold of 0.01 were used. Viscosity and refractive index of PBS at 25°C were used for data analysis.