Protein Crystallography and Biophysics Centre (BiophysX)
Institute of Structural and Molecular Biology (ISMB)
Birkbeck College / University College London

(Agilent 1100, Wyatt DAWN 8+, Wyatt Optilab T-rex)

Size Exclusion Chromatography coupled with Multi-Anlge Static Light Scattering (SEC-MALS) provides direct information on the molecular weight of the sample, so it can identify aggregates or oligomerization, characterize large complexes and generally assess the homogeneity of a sample. Using in addition UV detector, each species can be dissected into different components, particularly glycosylation or bound detergents. SEC-MALS is particularly powerful for optimizing the detergent content on membrane proteins as also for studying protein/DNA/RNA complexes and glycosylated proteins.

Set up and Sample Requirements
  • A Size Exclusion column usually of a 10/300 size is ideal for the current set up. A 3.2/300 column can also provide excellent results if the sample quantity is low
  • Volume: typically 100 μl for a 10/300 column. The injector has a range 0.1 - 100 μl therefore the injection volume can be adjusted depending on the actual parameters for each experiment (Volumes lower than 20 μl are not recommended though).
  • Concentrations: For the 10/300 columns 1 mg/ml is a reasonable approximation. 10-fold lower concentrations have worked for the 2.3/300 column.
  • Sample purity: purity is very important, since aggregates or other impurities can affect significantly the signal.
  • Buffers need to be freshly made, filtered and degassed.
  • A fraction collector is installed as default in the system.
Wyatt bibliography

Please contact us for detailed protocols and planning your experiment.

ISMB Protein Crystallography and Biophysics Centre, Birkbeck, University of London
Last modified April 2021