Small Angle X-ray Scattering (SAXS)

We performed small-angle X-ray scattering (SAXS) experiments at the SIBYLS beamline 12.3.1 at the Advanced Light Source. This beamlne has additional inline instrumentation and detectors coupled to a size exclusion column^1,2. XXX μl of XXX mg ml^-1 sample was suspended in XXX SEC running buffer. We set the X-ray wavelength to 1.127 Å and the sample-to-detector distance to 2,100 mm. This combination gives scattering vectors (q) ranging from 0.01 Å^-1 to 0.4 Å^-1. The scattering vector is q = 4πsinθ/λ, where 2θ is the scattering angle. We coupled the SAXS flow cell to an Agilent 1260 Infinity HPLC system using a Shodex XXX SEC column equilibrated with the running buffer as indicated above with a flow rate of 0.65 ml min^-1.

The eluent was subject to the following: 1) ultra-violet light (UV) at 280 nm, 2) multi-angle light scattering (MALS), 3) quasi-elastic light scattering (QELS), 4) SAXS, and 5) a refractometer. For SAXS measurements, 2 sec X-ray exposures were collected continuously during a 25 min elution. All frames for analyses had one SAXS frame corresponding to the running buffer before the detection of a peak subtracted from each.

The radius of gyration (Rg) was calculated for each of the subtracted frames using the Guinier approximation: I(q) = I(0) exp(−q²Rg²/3) with the limits qRg < 1.3. The elution peak was compared to the integral of ratios to background and Rg relative to the recorded frame using the program RAW³. Uniform Rg values across an elution peak represent a homogeneous sample. Final merged SAXS profiles, derived by integrating multiple frames at the elution peak, were used for further analyses. We calculated the Guinier plot to provide information on the aggregation state, the volume of correlation (Vc) to estimate the molecular weight⁴, and the pair distribution function [P(r)] to calculate the maximal inter-particle dimension⁵.

MALS experiments used an 18-angle DAWN HELEOS II light scattering detector connected with an Optilab refractive index concentration detector (Wyatt). A 55 μl sample of 7 mg ml-1 BSA monomer in the buffer noted above, and a refractive index increment (dn/dc) value of 0.18, was used for system normalization and calibration. We used light scattering experiments to perform analytical scale chromatographic separations for MW determination of the principal peaks in the SEC analysis. UV, MALS, and the differential refractive index data were analyzed using the Wyatt Astra 8 software package to monitor the homogeneity of the sample across the elution peak, which complements the SEC-SAXS signal validation.