Superdex s200 5 150 gl increase column

SEC-MALS and refractometry were performed on a Superdex S200 5/150 GL increase column (GE Healthcare). Twenty-five microliters of AdhE mutant protein were injected at a concentration of 10 mg ml⁻¹ in buffer 0.05 M Tris-HCl pH 7.5, 0.2 M NaCl. On-line MALS detection was performed with a miniDAWN-TREOS detector (Wyatt Technology Corp., Santa Barbara, CA) using a laser emitting at 690 nm and by refractive index measurement using an Optilab T-rex system (Wyatt Technology Corp., Santa Barbara, CA). Weight averaged molar masses were calculated using the ASTRA software (Wyatt Technology Corp., Santa Barbara, CA).

Size exclusion chromatography (SEC) experiments coupled to MALS and refractometry were performed on a Superdex S200 5/150 GL increase column (GE Healthcare). 25 μl of IbpS protein were injected at a concentration of 10 mg ml^-1 in 50 mm Tris-Cl pH 7.0, 100 mm NaCl. On-line MALS measurement was carried out with a miniDAWN-TREOS detector (Wyatt Technology Corp., Santa Barbara, CA) using a laser emitting at 690 nm and by refractive index measurement using an Optilab T-rex system (Wyatt Technology Corp., Santa Barbara, CA). Weight averaged molar masses (Mw) were calculated using the ASTRA software (Wyatt Technology Corp., Santa Barbara, CA).

Multi-angle laser light scattering (MALS) and small-angle x-ray scattering (SAXS) were used in conjunction with size-exclusion chromatography (SEC) to assess the molecular mass of RPV1^TIR20-193 in solution. SEC-SAXS was performed at the SAXS/WAXS beamline of the Australian Synchrotron. A Pilatus 1M detector at a sample-to-detector distance of 1.6 m and an energy of 12 keV yielded data over a q-range of 0.007–0.361 Å^-1, where q=4π.sin (θ)/λ. 1 mg of both RPV1^TIR and RPV1^TIR20-193H34A were separated over an inline 3 mL Superdex S200 5/150 GL Increase column (GE Healthcare) at 16°C, at a flow rate of 0.2 mL/min in 10 mM HEPES (pH 7.5), 150 mM NaCl buffer with 1 mM DTT. Frames were collected in 2 s exposures. Data reduction and subtraction was performed using scatterBrain¹. 100 frames immediately preceding each peak were summed and normalized to obtain buffer blanks, which were subtracted from each individual frame across the peak. Values of R_g and I(0) were calculated from each frame via Autorg in the PRIMUS suite (Petoukhov et al., 2012 (link)), and molecular masses were calculated from the volume of correlation for points where 0 < q < 0.3 Å^-1 (Rambo and Tainer, 2013 (link)).