Superdex 200 increase 3.2 300 sec column

Analysis of JMJD7 was performed using an Agilent 1260 HPLC with a Superdex 200 Increase 3.2/300 SEC column (GE Healthcare) coupled to a DAWN HELEOS II scattering detector (Wyatt Technologies) and Optilab T-rEX (Wyatt Technologies) differential refractive index detector. The MALS detector was equilibrated overnight in running buffer to ensure minimal background light scattering. ~30 μg of protein was loaded on-column (column compartment was set to 20°C) at a flow rate of 75 µL/min in 50 mM HEPES, 200 mM NaCl, 2% glycerol, pH 7.5. Data acquisition and analysis were performed with Astra 6.1 software (Wyatt Technologies).
CD spectra were acquired using a Chirascan CD spectrometer (Applied Photophysics model) equipped with a Peltier temperature-control cell holder. Experiments were performed at 20°C in a 0.1 cm pathlength cuvette using 0.2 mg/mL of protein in 10 mM sodium phosphate buffer, pH 8.0. Data were recorded in triplicate from 260 to 185 nm in 0.5 nm steps with a 1 nm bandwidth. Spectral data were baseline corrected, averaged, and smoothed using the Savitzky-Golay filter (smooth = 8).