4.7. Sedimentation Velocity Analytical Ultra-Centrifugation

Sedimentation velocity analytical ultra-centrifugation experiment was done using a ProteomeLab XL-A centrifuge (Beckman Coulter, Brea, California, USA) equipped with An-50 Ti analytical 8-place titanium rotor. The sectors of each Epon double-sector centerpieces were filled with 400 µL of SEC buffer and 390 µL of the protein sample, respectively. The AtNRP2 and AtNRP2NT/CT samples were diluted in their SEC buffer to obtain an OD₂₈₀ of 0.5. The samples were centrifuged at 40,000 rpm, at a temperature of 20 °C and 350 frames were collected. Absorbance data were acquired at a wavelength of 280 nm by taking two averages per scan. Absorbance scans were taken at an interval of 1 min. Buffer viscosity, protein partial specific volume, and viscosity were calculated using the software SEDFIT [68], and data analysis were performed with SEDFIT [68] and SEDPHAT using c(s) distribution analysis [69]. C (s) distribution plots were made using GUSSI.