Coupling of Size-Exclusion Chromatography and Small-Angle X-ray Scattering – Principles and Applications

Biomolecular interactions are important for their biological function and SAXS can provide key structural information to decipher these functions. However, aggregation or formation of complex oligomeric mixtures complicates analysis of SAXS data. Coupling of size exclusion chromatography and SAXS provides an efficient tool for screening and structural studies of (challenging) biomolecular samples in the home lab.


Small-angle X-ray scattering (SAXS) techniques can provide structural and dynamic information for biological macromolecules such as proteins, nucleic acids and their complexes in solution under near-native conditions.[1,2] The prerequisite for accurate interpretation of solution SAXS data is that the macromolecule of interest is monodisperse, and that any interparticle interactions such as aggregation or repulsion are negligible. These requirements are typically checked by careful analysis of the SAXS data or using other suitable techniques, such as dynamic light scattering. Several SAXS-derived parameters such as the (concentration-dependent) molecular mass and the Guinier plot inform on the monodispersity of the sample.

Biomolecular interactions are important characteristics of biomolecules

Biomolecular interactions can be specific or unspecific. Proteins, for example, can exist in biologically rele-vant oligomeric or aggregate states. On the other hand, proteins can form large, unspecific aggregates during protein expression and purification steps. Even if present only in small amounts, these aggregates distort the SAXS curve because the forward scattering intensity of a particle depends on the square of its volume.

Therefore, and to enable structural analysis, the time between protein purification and structural analysis using SAXS is desired to be kept as short as possible.


1. Madl, T. G., F. Sattler, M., NMR and small-angle scattering-based structural analysis of protein complexes in solution. J. Struct. Biol. 2011, 173, 472-482.

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

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