HPLC

Small-angle X-ray scattering (SAXS) can provide accurate structural information and low-resolution shapes of macromolecules in solution. Often, it is not possible to obtain a stable monodispersed sample of the protein of interest required for a successful SAXS experiment, in particular for multi-subunit protein complexes. In these circumstances, size exclusion chromatography (SEC) coupled with SAXS can facilitate the separation of monodispersed protein from a polydispersed sample for a sufficient amount of time to collect useful SAXS data.

New SEC system in-situ at the BM29 beamline, to be used in parallel with the sample changer robot, is available for external users from June 2015. The new system (Shimadzu) replaces the old one (Malvern) used and has improved characteristics: auto sampler allowing sample loading without loses (smallest sample quantity needed 10microL), buffer gradient possible (up to 4 components) and conductivity measurement is included. Figure shows the actual SEC set-up (white boxes on the right part of the photograph behind the display) installed on the experimental marble table in the BM29 experimental hutch. The particular attention was made to keep as short as possible the tubing between the column end and the quartz capillary where samples are exposed to X-rays to minimise dilution. The switch between two modes (robot and SEC) is quick and fully automatised.