Why are my large proteins eluting before my small ones on a size exclusion column – shouldn the smaller ones just slide right through?
SEC relies on the volume a molecule can travel through. The more volume it has to travel through, the longer it will take to elute. SEC resins have tunnels of various sizes, the range of which are given by the manufacturer for theoretical, globular proteins. Small proteins can access a large amount of these tunnels and take a longer time to come out, while larger ones in extreme cases do not even enter any of the beads and snake their way through the beads. Thus, they see little of the total volume and take less time to elute from your column.
