Mario Lebendiker, PhD, Head, Protein Purification Facility, Wolfson Center for Applied Structural Biology, Hebrew University Jerusalem
MALS coupled with size exclusion chromatography (SEC-MALS) is a standard and common method for characterizing protein mass, shape, aggregation, oligomerization, interactions, and purity. The limited resolution of SEC interferes in some cases with the accurate analysis that can be achieved by MALS. These include mixtures of protein populations with identical or very similar masses, oligomers with poor separation and small peptides. We recently developed a new technology that combines MALS with protein separation according to charge, IEX (IEX-MALS). The high-resolution separation technique IEX (IEX-MALS) allows a precise analysis of samples that cannot be resolved (or have limited analysis) by SEC-MALS. The technology is very simple, and unlike SEC-MALS that needs prolonged column equilibration, here the equilibration is performed in a few minutes, and the injection volume is not restricted. We will discuss advantages and disadvantages of IEX-MALS vs. SEC-MALS, as well as its applicability at low scale during processing development, allowing a fast on-line identification of the target with the correct oligomeric conformation, from other conformations or other proteins. Its applicability in membrane proteins purification, characterization, and separation from free micelles, and many other possible applications will also be presented.