Superdex 75 increase 10 300 gl size exclusion column

The ¹⁵N-labeled JD and CTD were exchanged into 20 mM Tris 100 mM NaCl 1 mM DTT pH 7 buffer using a Superdex 75 Increase 10/300 GL size-exclusion column (GE) in preparation for solution NMR. Each HSQC (heteronuclear single quantum coherence) run was performed with 50 μM ⁵N-labeled JD for 4 h at 1 scans/min with the temperature fixed at 299 K. After each run, unlabeled CTD was titrated into the sample at 2× (100 μM) the JD concentration, following serial dilutions of 1× (50 μM), 0.5× (25 μM), 0.25× (12.5 μM), and 0.125× (6.125 μM) sequentially. All scans were collected on an Agilent DD2 600 MHz instrument outfitted with a cold probe at the UT Southwestern Biomolecular NMR Facility. Each spectrum was converted into a readable format and phase corrected using NMRPipe^{60 (link)}. Peak assignments were based on the deposited information from BMRB (11417). The software Sparky^{59 ,61 (link)} was used to analyze the peak shifts across all spectra.

Purified His-tagged proteins EH1.1 (1 mg/ml) or EH1.2 (2 mg/ml) were injected onto a Superdex 75 Increase 10/300 GL size exclusion column (GE Healthcare), equilibrated with 20 mM HEPES pH 7.4, 300 mM NaCl, coupled to an online UV-detector (Shimadzu), a mini DAWN TREOS (Wyatt) multi-angle laser light scattering detector and an Optilab T-rEX refractometer (Wyatt) at room temperature. A refractive index increment (dn/dc) value of 0.185 ml/g was used. Band broadening corrections were applied using parameters derived from BSA injected under identical running conditions. Data analysis was carried out using the ASTRA6.1 software.