High performance liquid chromatography pump

The sizes of FL MexT and MexT RD were assayed using SEC-MALS. A high-performance liquid chromatography pump (Agilent, USA) was connected to a Superdex-200 10/300 GL gel filtration column (GE Healthcare) and a MALS instrument (Wyatt Dawn Heleos, USA). The size-exclusion chromatography column was pre-equilibrated with buffer comprising 20 mM Tris-HCl (pH 8.5), 500 mM NaCl, and 2 mM 2-mercaptoethanol for FL MexT; and 20 mM Tris-HCl (pH 8.5), 300 mM NaCl, and 2 mM 2-mercaptoethanol for MexT RD. Bovine serum albumin (2 mg/ml) was used as the standard. MexT RD and FL MexT samples (2 mg/ml) were injected onto the column and eluted at a flow rate of 0.2 ml/min. The datasets were evaluated using the Debye model for fitting static light-scattering data, and refractive index peaks were presented in EASI graphs created using Astra V software (Wyatt Dawn Heleos).

SEC-MALS experiments were performed using a High-performance liquid chromatography pump (Agilent) connected to a Superdex-200 10/300 GL (GE Healthcare) gel filtration column and a Wyatt DAWN HELIOS MALS instrument. The gel filtration column was pre-equilibrated with 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM β-mercaptoethanol. Bovine serum albumin at 2 mg/mL was used as a protein standard. The VV2_1132 protein sample at 3 mg/mL was injected into the column and eluted at a flow rate of 0.2 ml/min. The data were evaluated using the debye model for static light scattering data fitting and represented using an EASI graph with a RI peak in the ASTRA V software (Wyatt).