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Chromatography-Coupled SAXS

Biological small-angle X-ray scattering experiments can be performed in-line with liquid chromatography. In these experiments, protein sample is injected onto a protein purification column. Typically, size-exclusion chromatography is used, as it has a constant buffer composition that does not complicate future buffer subtraction. The eluting protein travels immediately into the X-ray beam. A major benefit of this technique is that it removes aggregation from samples. Small-angle scattering is extremely sensitive to large species in solution because X-ray scattering intensity scales proportionally with the square of a molecule's mass. In a non-homogenous protein sample, aggregates will often dominate the signal. By separating out aggregated species immediately prior to entrance into the SAXS beam, this problem is partially alleviated. Chromatography-coupled SAXS can also be used to separate protein species in a mixture, such as different oligomeric states. However, this technique can be limited by chromatography resolution as well as possible interconversion of species during and after elution.