Multi-angle Light Scattering Detectors

A new application note details how Wyatt Technology’s DAWN HELEOS Multi-Angle Light Scattering (MALS) instrument to track changes in proteins as salt concentrations and buffer solutions were modified. For this application, the 18-angle instrument was coupled to a UV detector and Optilab rEX refractive index detector (Wyatt Technology). A TSKgel G3000SW column from Tosoh Bioscience was used while the analysis was performed within a 30 minute run time at a 1 ml/min flow rate. A PBS isocratic mobile phase was employed. Prior to loading onto the HPLC column, the protein was thoroughly dialyzed into various buffers containing different concentrations of salt spanning 150 mM to 500 mM as well as different buffer systems with pH values ranging from 5 to 8.5. The different states of the protein were separated into multiple peaks through SEC-HPLC and the molecular weights for these peaks were simultaneously determined by MALS. The molecular weights detected by MALS correlated very well with those expected for monomer, dimer, trimer and higher oligomers of the protein. MALS detection revealed that in a couple of buffers the molecular weights were lower or higher than those expected, suggesting salt/pH dependent dissociation or association of the protein subunits.