Protein titration curves by combined isoelectric focusing-electrophoresis with hemoglobin mutants as models

By performing electrphoresis perpendicular to a stationary pH gradient generated by focused carrier ampholytes in a gel slab, a pictorial representation of a protein titration curve is obtained. By running a protein and its genetic mutants in parallel, the shape of the relative titration curve reveals which charged amino acid has been substituted. Within a given family of proteins, under a constant set of experimental conditions, the relative mobility at any given pH can be correlated with the number of protons lost or acquired by the protein.