Minidawn treos light scattering detector

Purified Ino80 CTD was used for molecular weight analysis by size exclusion chromatography-multi-angle light scattering (SEC-MALS) with a Superdex 75 (GE Healthcare) size exclusion column. The column was extensively equilibrated in ‘MALS Buffer’ [25 mM HEPES, 300 mM NaCl] at room temperature before applying a 100 µL of Ino80 CTD through a capillary loop. The system flow was maintained at 0.5 mL/min. Light scattering and UV absorbance data at 280 nm were collected across a 25 mL volume post-injection using a miniDAWN TREOS light scattering detector (Wyatt). All data were exported and re-plotted in GraphPad Prism.

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.

SEC-MALLS measurements were run at 0.5 ml/min over an S-200 Superdex 10/300 column (GE Healtcare) in 20 mM TRIS pH 7.4, 150 mM NaCl with a Schimadzu HPLC system and MiniDAWN TREOS light scattering detector and Optilab rEX refractive index detector (Wyatt Technology Corporation). Data was analysed with ASTRA 6 software (Wyatt Technology Corporation).