SEC-MALS Analysis of Protein Oligomers

SEC-MALS analysis for molecular weight and oligomer state determination was performed as described [35 (link)]. Briefly, TvCyP2 (1 mg/mL) and TvCyP2-∆N (1 mg/mL) were used for SEC-MALS analysis. The column (Enrich Tm SEC. 70 10 × 300, Bio-Rad Laboratories, Santa Barbara, CA, USA) was used with a flow rate of 0.5 mL/min in the buffer system of 20 mM Bis-Tris, 50 mM NaCl at pH 6.0 and 25 °C. Detectors such as an ultraviolet-visible (UV) detector (QELS, Wyatt Technology, Santa Barbara, CA, USA), a static light-scattering detector (mini DAWN TREOS, Wyatt Technology, Santa Barbara, CA, USA), a quasi-elastic light-scattering detector (QELS, Wyatt Technology, Santa Barbara, CA, USA), and a refractive index detector (Optilab T-rEX, Wyatt Technology, Santa Barbara, CA, USA) all were aligned with the column. Bovine serum albumin (Sigma, A1900, Saint Louis, MO, USA) was used as a standard for calibration and optimization. The molecular weight was calculated by using ASTRA 6 with dn/dc value set to 0.185 mL g^-1.