In-line SEC-SAXS of HSA Protein Using a Laboratory SAXS Instrument

Small-angle X-ray scattering is a widely used technique in structural biology for the determination of protein size and shape. Biological macromolecules in solution can exist in numerous conformations and generally have a strong tendency for agglomeration, making data evaluation of such molecules without prior purifications sometimes tedious. Size-exclusion chromatography (SEC) is capable of separating molecules by size. In-line SEC in combination with SAXS can considerably improve data quality and thus the accuracy of the data evaluation.


Small-angle X-ray scattering (SAXS) is a commonly used method for the investigation of macromolecular samples, e. g. in biochemistry.[1] SAXS measurements have seen a remarkable growth over the last years due to improvements in beam delivery (3rd and 4th generation synchrotrons, modern laboratory X-ray sources), detection technology, and advances in data analysis. Experiments that were only possible at synchrotron facilities a decade ago now become available with laboratory instruments.

In a typical SAXS experiment, billions of nanostructures (molecules or particles) are probed for their scattering signal. In the case of macromolecules these can exist in many conformational states such as monomeric and oligomeric species, different folding states, etc. This can make data evaluation in SAXS experiments challenging and the resulting information quality of ab initio modeling computations will be low.2 Purification of a sample solution prior to an experiment can facilitate and increase the accuracy of data evaluation drastically and is – especially in the evaluation of protein molecules – an essential step for sample preparation.

Size-exclusion chromatography (SEC) is an established preparation method at SAXS beamlines in modern synchrotron facilities. In SEC, a mixture of differently sized molecules (e. g., monomers, dimers, etc.) is purified to obtain monodisperse fractions. The underlying principle is that macromolecules of different sizes will have different retention times on a porous chromatographic resin. Thus, large molecules will elute faster than smaller ones, separating the individual fractions by size. SEC experiments are commonly monitored using UV absorption measurements to determine at which time which fraction elutes.Due to the nature of the separation process the individual fractions are very low in concentration. Hence good quality SAXS data requires high power X-ray sources and/or long exposure times. Thus, in the past the SEC-SAXS method has been limited to synchrotron beamlines. Recent advances in laboratory instrumentation (e. g., powerful SAXS instruments based on metaljet or high power sealed tube sources) make this method now also accessible to the home lab, expanding the possibilities for both research and routine measurements. Here we show combined SEC-SAXS experiments carried out on the Anton Paar SAXSpace instrument on a HSA protein sample to illustrate the potential of performing SEC-SAXS measurements also on laboratory instruments.


1. Schnablegger, H.; Singh, Y., The SAXS Guide - Getting acquainted with the principles. 4th ed.; Anton Paar GmbH: Graz, Austria, 2017.

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