Heleos 2 18

Hundred microlitres of DIX samples were resolved on a Superdex-75 HR10/300 analytical gel filtration column (GE Healthcare) at 0.5 ml min⁻¹ in 20 mM Tris (pH 7.4), 200 mM NaCl, 0.01% (w/v) NaN₃ before light scattering (on a Wyatt Heleos II 18 angle light scattering instrument coupled to a Wyatt Optilab rEX online refractive index detector) in a standard SEC-MALS format. Heleos detector 12 was replaced with a Wyatt's QELS detector for dynamic light scattering measurements. Protein concentration was determined from the excess differential refractive index based on 0.186 RI increment for 1 g ml⁻¹ protein solution. Concentrations and observed scattered intensities at each point in the chromatograms were used to calculate the radius of gyration and absolute molecular mass from the slope and the intercept of the Debye plot, using Zimm's model as implemented in Wyatt's ASTRA software. Autocorrelation analysis of data from the dynamic light scattering detector was also performed using Wyatt's ASTRA software, and the translational diffusion coefficients determined were used to calculate the hydrodynamic radius using the Stokes-Einstein equation.

SEC-MALS measurements were performed at room temperature using a Wyatt Heleos II 18 angle light scattering instrument coupled to a Wyatt Optilab rEX online refractive index detector. Samples of DRp53(302-331) and DRp53(302-345) in 50 mM Tris (pH 7.5) and 150 mM NaCl were resolved on a Superdex 75 10/300 analytical gel filtration column at a flow rate of 0.5 ml/min and passed through the light-scattering and refractive index detectors in a standard SEC-MALS format. Data analysis was performed using the ASTRA5 software (Wyatt Technology), with bovine serum albumin as a calibration standard.

100 μl SSDP, 6xHis-MBP-Chip–6xHis-Lip-SSDP or 6xHis-MBP-LDB1–6xHis-Lip-SSDP samples were resolved on a Superdex S-200 or Superose 6 HR 10/300 analytical gel filtration column (GE Healthcare) at 0.5 ml min⁻¹ in 25 mM phosphate buffer, 150 mM NaCl, pH 6.7 before light scattering and concentration determination using refractive index (RI) or UV absorbance in a standard SEC-MALS configuration (containing a Wyatt Heleos II 18 angle light scattering instrument coupled to a Wyatt Optilab rEX online RI detector). Protein concentration was determined from the excess differential refractive index based on 0.186 RI increment for 1 g ml⁻¹ protein solution. Concentrations and observed scattered intensities at each point in the chromatograms were used to calculate absolute molecular mass from the intercept of the Debye plot, using Zimm's model as implemented in Wyatt's ASTRA software. The stoichiometries of ChiLS indicated by the model-free RI measurements (using dn/dc 0.186 for 1g ml⁻¹ protein) were further confirmed by using appropriate UV extinction coefficients and UV absorbance as the concentration measurement, which produced essentially identical masses to those from RI. They were independent of protein concentration (in the range of 0.1–10 mg ml⁻¹).