Size-Exclusion Chromatography with Multi-Angle Light Scattering for Protein Oligomers
Transcription
A protein oligomer is a complex of multiple protein subunits that are bound together to form a larger functional unit.
To determine the molecular weight of protein monomers and their oligomers in solution by SEC-MALS, a combination of size-exclusion chromatography and multi-angle light scattering, begin with a size-exclusion chromatography, or SEC, column.
The column contains porous sieve-like spherical gel beads that trap smaller-sized molecules within them. Equilibrate the column with a buffer compatible with the protein's native state.
Load a sample containing protein monomers and oligomers at the top of the column.
During the flow, the oligomers pass through the interparticle space of the column, while the monomers become entrapped within the pores of the beads. This causes the larger-sized protein oligomers to travel faster than the smaller-sized monomers.
Direct the fast-eluting oligomers, followed by the slow-eluting monomers, to the MALS detector.
As they pass through the detector, a laser light illuminates them, which is scattered at different angles. The multi-subunit oligomers scatter more light than the monomer.
The intensity of the scattered light at multiple angles is directly proportional to the molecular weight of the protein oligomer and monomer.
