Optilab refractive index detector

To assess changes in the oligomeric state of TsaC, in-line SEC–MALS and DLS experiments were conducted. SEC–MALS experiments were conducted using an Agilent 1260 Infinity II HPLC equipped with a 4 °C chilled multisampler and an AdvanceBio 300A 2.7 μm 4.6 × 300 mm (Agilent) SEC column. The SEC–MALS column was pre-equilibrated in buffer consistent with the individual experimental condition (50 mM Hepes [pH = 8.2], 200 mM NaCl, 5% [v/v] glycerol, and either NAD⁺ or NADH). The experiment was initiated with a 15 μl injection of 90 μM protein, and the column was run at room temperature and 0.25 ml/min. SEC–MALS data were collected as the protein migrated out of the SEC column on a Wyatt Neon DAWN ambient and analyzed using Astra Software, version 8.2. Concentrations were measured using both a 1260 Infinity II multiwavelength detector monitoring absorbance at 280 nm and the OptiLab Refractive Index Detector (Wyatt). DLS experiments were conducted with 5 μM to 1 mM enzyme, 1 mM to 2 mM coenzyme, and 1 mM to 2 mM substrate–product as indicated. DLS data were collected at 25 °C using a Wyatt microvolume disposable cuvette on a Wyatt DynaPro NanoStar II, with three data segments collected per sample with a 1-min incubation between each segment. DLS data were analyzed using Dynamics Software, version 8.2 (Waters, Wyatt Technology).

The SEC-MALS analysis of the purified proteins (in SEC buffer) was carried out using a miniDAWN detector from Wyatt Technologies in on-line mode (connected to the Shimadzu HPLC system). Before entering the MALS detector, the sample flows through an Optilab refractive index detector from Wyatt Technologies. This is used to measure the concentration of the protein sample. Molecular mass and polydispersity measurements were carried out using the ASTRA software from Wyatt Technologies. About 30 to 50 μl of the purified (and frozen-thawed) sample was filtered and loaded with flow rate 500 μl/min onto the Superdex 200 Increase 10/300 GL column pre-equilibrated with SEC buffer (0.1 μm filtered and degassed).

Recombinant, monoclonal anti-TNFα antibody (prepared in house) and soluble, trimeric TNFα (R&D Systems, Minneapolis, MN, USA) were mixed in a 3:1 molar ratio to form complexes composed of three anti-TNFα molecules binding one TNFα trimer. The mixtures were incubated for 24 or 48 h at 37 °C. The average molecular weights of the TNFα immune complexes were measured using a Size Exclusion Chromatography (SEC) system coupled with Multi-Angle Light Scattering (MALS) and Refractive Index (RI) detection. HPLC data were collected on an Agilent 1100 liquid chromatograph (Palo Alto, CA, USA) equipped with an OptiLab refractive index detector (Wyatt Technology, Santa Barbara, CA, USA) and a MiniDawn TREOS light scattering detector (Wyatt Technology, Santa Barbara, CA, USA). A Bio SEC-5 (Agilent, Palo Alto, CA, USA) size exclusion column (4.6 × 300 mm, 5 μm) was used for the analysis. An isocratic mobile phase of 0.15 M sodium phosphate (pH 7.0) at a flow rate of 0.35 mL/min was employed for the separation at an ambient column temperature. An injection volume of 25 µL was employed. Data were processed using both Empower 2 software (Build 2154, Waters, Milford, MA, USA) and ASTRA software (Version 5.3.4, Wyatt Technology, Santa Barbara, CA, USA). Each sample (monomer or complex) was analyzed directly post sample preparation.