Practice question

Proteins encoded by single gene often exist as multiple isoforms created by alternative splicing, phosphorylation, acetylation, deamidation or limited proteolysis, producing subtle variations in charge and mass indistinguishable in one-dimensional systems. SDS-PAGE masks charge differences and poorly resolves small mass changes, while isoelectric focusing alone cannot separate mass variants. Two-dimensional electrophoresis combining isoelectric focusing for pI separation and SDS-PAGE for molecular mass provides orthogonal resolving power, displaying isoforms as distinct spots across pH and size axes. This reveals charge variants and post-translational modifications co-migrating in single dimension, essential for proteomic isoform analysis.