Akta purifier upc10 fplc protein purification system

Protein molecular weights were determined by static light scattering (SLS) using a Wyatt Dawn Heleos II multi-angle light scattering detector (Wyatt Technology) coupled to an AKTA Purifier UPC10 FPLC protein purification system and a WTC-030S5 size-exclusion column (Wyatt Technologies). 100 µL TcVSP1 (2.5 mg/mL), ΔN1-TcVSP1 (2.4 mg/mL), ΔN2-TcVSP1 (1.9 mg/mL), and TbVSP1 (4.7 mg/mL) were subjected to SEC-MALS analysis individually with a running buffer containing 25 mM Tris (pH 7.5), 150 mM NaCl and 0.02% NaN₃ at a flow rate of 0.5 mL/min. 0.1 mM CaCl₂ (final concentration) was added into the same running buffer to investigate the effects of Ca²⁺. BSA (2 mg/mL) was used for system calibration. The absolute molecular weights of the individual peaks observed in the size-exclusion chromatograms were determined by SLS in conjunction with their corresponding refractive indices using an online refractometer connected downstream from the SLS detector (Wyatt Optilab rEX). A standard value of the refractive index, dn/dc = 0.185 mL/g, was used for all proteins. The buffer viscosity, η= 1.0226 cP at 25 °C was calculated by using SEDNTERP(31 ). The reference refractive index, 1.3459 RIU, was obtained from the running buffer that passed through the reference cell.