Minidawn treos multi angle light scattering detector

SEC analysis was performed on neurofascin 155^Ig1–6 HM and CG wt, and Thr216Ala HM samples to characterize monomer dimer exchange through peak shift. Purified samples (1–100 μM) were injected onto a Superdex200 10/300 increase (GE Healthcare) column equilibrated in SEC buffer and separated with a flow rate of 0.75 ml min⁻¹. For molecular weight characterization of neurofascin 155^Ig1–6 HM and Thr216Ala HM samples, light scattering measurements were performed using a miniDAWN TREOS multi-angle light scattering detector (Wyatt), connected to a differential refractive index monitor (Shimadzu, RID-10A) used for protein concentration quantification. Collected chromatograms were analyzed and processed using ASTRA6 software (Wyatt, using a calculated dn/dc value of 0.182 ml g⁻¹, determined from dn/dc of 0.188 and 0.145 for the protein and glycan parts respectively, and 8% glycosylation estimated from crystallographically confirmed glycosylation sites). Instrument calibration was assessed by injection of 5 mg ml⁻¹ monomeric conalbumin (Sigma-Aldrich), using in this case a dn/dc value of 0.185 ml g⁻¹.

For protein molar mass determination, purified SHLD3s–REV7, SHLD3 (1 to 58)–REV7, and SHLD2.3–REV7 proteins were analyzed using an ÄKTA-MALS system. A mini DAWN TREOS multiangle light scattering detector (Wyatt Technology) and an Optilab T-rEX refractometer (Wyatt Technology) were used in-line with a Superdex200 10/300 gel filtration column (GE Healthcare) preequilibrated in the buffer (20 mM Tris⋅HCl [pH 7.5], 150 mM NaCl, and 2 mM DTT) at a flow rate of 0.2 mL/min. Separation and ultraviolet (UV) detection were performed by ÄKTA Pure System (GE Healthcare), light scattering was monitored by the mini DAWN TREOS system, and concentration was measured by the Optilab TrEX differential refractometer. Molar masses of proteins were calculated using the Astra 6.1 program (Wyatt Technology) with a dn/dc value (refractive index increment) of 0.185 mL/g. The data were plotted using Prime8 software (GraphPad).

A miniDAWN TREOS multi-angle light scattering detector, with three detector angles (43.6°, 90° and 136.4°) and a 658.9 nm laser beam (Wyatt Technology, Santa Barbara, CA), with a Wyatt QELS dynamic light scattering module for determination of hydrodynamic radius and an Optilab T-rEX refractometer (Wyatt Technology), were used in-line with a size exclusion chromatography analytical column, Superdex 200 Increase 10/300 GL (GE, Life Science, Marlborough, MA) equilibrated in buffer (50 mM tris, 150 mM NaCl and 4 mM MgCl₂ [pH 8.0]).
Experiments were performed using an AKTA explorer system with a UV-900 detector (GE), at 0.8 ml/min. All experiments were performed at RT (25°C).
Data collection and mass calculation by SEC-MALS analysis were performed with ASTRA 6.1 software (Wyatt Technology). The refractive index of the solvent was defined as 1.331 and the viscosity was defined as 0.8945 cP (common parameters for PBS buffer at 658.9 nm). dn/dc (refractive index increment) value for all samples was defined as 0.185 mL/g (a standard value for proteins). For the SIRT6 experiment, 150 ul 4.5 mg/ml human-SIRT6-His was injected. For SIRT6+DNA, 200 μl human-SIRT6-His + 50 ul DNA was injected after 1 hr incubation at 37°C.