Human milk proteins: Separation of whey proteins and their analysis by polyacrylamide gel electrophoresis, fast protein liquid chromatography (FPLC) gel filtration, and anion-exchange chromatography

Human milk proteins are of nutritional and physiological significance to the newborn infant. To further study these proteins, a rapid procedure to separate an analyze human milk whey proteins was developed using fast protein liquid chromatography (FPLC). First, to separate whey proteins from casein, different variables such as low- or high-speed centrifugation at different temperatures with or without adjustment of pH to 4.6 or 4.3 and with or without addition of calcium to whole milk or skim milk were tested. Each variable was evaluated by gel filtration, anion-exchange chromatography, sodium dodecyl sulphate (SDS)-polyacrylamide gradient gel electrophoresis, immunoelectrophoresis, and immunodiffusion. The optimum method for a discrete separation of whey and casein is the adjustment of whole milk to pH 4.3 with addition of 60 mmol calcium/L, followed by ultracentrifugation. Rapid and sensitive separation and analysis of whey proteins was achieved by FPLC gel filtration and anion-exchange chromatography.