Which method is primarily used to separate proteins based on their size?

The method primarily used to separate proteins based on their size is gel filtration chromatography. This technique, also known as size-exclusion chromatography, utilizes a porous gel material as the stationary phase. When a protein mixture is applied to the column filled with this gel, smaller proteins can enter the pores of the beads, while larger proteins are excluded and elute from the column more quickly. Consequently, as the proteins move through the column, they are separated based on their size, with larger molecules eluting first and smaller molecules eluting later. This method is particularly effective for analyzing protein complexes and determining molecular weights.

In contrast, affinity chromatography is utilized for separating proteins based on their specific binding interactions with ligands, not size. Western blotting is a technique used for detecting specific proteins in a sample after they have been separated by gel electrophoresis, but it does not separate proteins based on size itself. Mass spectrometry, while a powerful tool for analyzing proteins and their properties, is mainly used for identification and quantification rather than separation by size.