Superdex 200 10 30 gl size exclusion column

DNA constructs for CLC-ec1 C85A/H234C (WT), and C85A/H234C/R230C/L249C (RCLC) have been described earlier, along with their expression and purification methods (4 (link), 6 (link)). Briefly, BL21-AI E. coli cells transformed with the expression plasmid were lysed by sonication and the protein extracted from membrane fragments into 2% n-decyl-β-D-maltopyranoside (DM; Anatrace) containing 5 mM TCEP [Tris (2-carboxyethyl) phosphine; Soltec Bioscience] to ensure that the introduced cysteine residue at residue 234 (H234C) remains in a reduced state for maleimide labeling. After pelleting cellular debris by centrifugation, the protein was affinity purified using TALON cobalt affinity resin (Clontech Laboratories) followed by size exclusion chromatography on a Superdex 200 10/30 GL size exclusion column (GE Healthcare) into size exclusion buffer (SEB): 150 mmol L^-1 (mM) NaCl, 20 mM MOPS pH 7.0, and 5 mM analytical-grade DM. Molar extinction coefficients for WT and RCLC are 46,020 M⁻¹ cm⁻¹ and 49,630 M⁻¹ cm⁻¹ respectively, and molecular weights for WT and RCLC are 52,000 g/mol and 49,630 g/mol respectively.

Size exclusion chromatography–multiangle light scattering (SEC-MALS) was performed as previously described (Klebba et al., 2015b ). Briefly, Asl mutants (50 μM, 100 μl) were injected onto a Superdex 200 10/30 GL size exclusion column (GE Healthcare Life Sciences) at 0.5 ml/min in buffer (25 mM HEPES, pH 7.5, 300 mM NaCl, 0.1% β-mercaptoethanol, and 0.2 g/l sodium azide). Eluent was analyzed by a tandem RI detector/Dawn Heleos II multiangle static light scattering (MALS) detector (Wyatt Technology). Light scattering and refractive index data were used to calculate the weight-averaged molar mass using Wyatt Astra V software (Wyatt Technology).