Size exclusion chromatography (SEC) coupled to Multi-Angle Static Light Scattering (MALS) separates molecular species within a sample by size and provides direct information on the molecular weight of each species. SEC-MALS can be used to identify complex formation and the stoichiometry of complexes or to simply assess the homogeneity of samples. Inline UV detection can be used to identify chemically distinct components such as glycosylation, nucleic acid and bound detergents. SEC-MALS is particularly powerful for providing information to enable optimization of detergent content of membrane proteins.
Wide-bore and narrow-bore SEC columns are available. The wide-bore column is optimal in terms of MALS signal.
What are the sample requirements?
- Samples should be free from aggregation and contain a small number of (almost) pure components (to enable good separation and identification of complexes)
- Concentration. For the wide-bore column 1mg/ml is a reasonable level. 10-fold lower concentrations have worked for the narrow-bore column.
- Volume is typically 100 μl for a sample. This can be reduced, but volumes lower than 20 μl are not recommended.
What other specific considerations are relevant?
- A wide range of buffers can be used, but any required buffer should not have substantial UV absorbance.
Partners offering this technique
MOSBRI reference partner sites for this technique: