Journal
JOURNAL OF APPLIED CRYSTALLOGRAPHY
Volume 51, Issue -, Pages 97-111Publisher
INT UNION CRYSTALLOGRAPHY
DOI: 10.1107/S1600576717017101
Keywords
size exclusion chromatography small-angle X-ray scattering; SEC-SAXS; coflow; SAXS; polydisperse proteins; molecular weight
Categories
Funding
- RSNZ Marsden fund
- NHMRC [1080784, 1045936, 1098290, APP1110971]
- National Health and Medical Research Council of Australia (NHMRC) RD Wright Fellowship [APP1105754]
- Cancer Council Victoria Postdoctoral Fellowship
- NHMRC IRIISS [9000220]
- Victoria Government
- NHMRC Programme [APP1071659]
- National Health and Medical Research Council of Australia [1080784, 1098290] Funding Source: NHMRC
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A new optimized size exclusion chromatography small-angle X-ray scattering (SEC-SAXS) system for biomolecular SAXS at the Australian Synchrotron SAXS/WAXS beamline has been developed. The compact configuration reduces sample dilution to maximize sensitivity. Coflow sample presentation allows an 11-fold increase in flux on sample without capillary fouling, improving throughput and data quality, which are now primarily limited by the full flux available on the beamline. Multi-wavelength fibre optic UV analysis in close proximity to the X-ray beam allows for accurate concentration determination for samples with known UV extinction coefficients and thus estimation of the molecular weight of the scattering particle from the forward X-ray scattering intensity. Fast-flow low-volume SEC columns provide sample throughput competitive with batch concentration series measurements, albeit with a concomitant reduction of potential resolution relative to lower flow rates and larger SEC columns. The performance of the system is demonstrated using a set of model proteins, and its utility to solve various challenges is illustrated with a diverse suite of protein samples. These developments increase the quality and rigor of SEC-SAXS analysis and open new avenues for biomolecular solution SEC-SAXS studies that have been challenged by low sample yields, temporal instability, radiation sensitivity and complex mixtures.
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