Highly sensitive periodic acid/Schiff detection of bovine milk glycoproteins electrotransferred after nondenaturing electrophoresis, urea electrophoresis, and isoelectric focusing

Abstract

Due to its lack of sensitivity, periodic acid/Schiff (PAS) staining of gels is not considered to be relevant for the detection of glycoproteins other than mucin-type glycoproteins after nondenaturing or urea polyacrylamide gel electrophoresis (PAGE). A simple and highly sensitive method was developed for the detection of bovine milk glycoproteins separated by nondenaturing PAGE, urea-PAGE, and isoelectric focusing. After electrophoresis, proteins were electrotransferred onto a polyvinylidene difluoride (PVDF) membrane and then glycoproteins were specifically stained by the Schiff's reagent after periodic acid oxidation. This method allowed the detection of the glycosylated forms of bovine α-lactalbumin with sufficient sensitivity after nondenaturing PAGE or isoelectric focusing of whey proteins. Combination of isoelectric focusing and PAS detection on a PVDF membrane provided a resolutive tool in the characterization of the different glycoforms of κ- casein and also of component PP3 of proteose peptones. Distinction between the non-glycosylated and glycoforms of κ-casein separated by urea-PAGE or isoelectric focusing was performed by com- paring Coomassie blue staining and PAS staining. Moreover, amino-terminal microsequencing of glycoproteins immobilized on the membrane could be performed after PAS staining. bovine milk protein / glycoprotein / electrophoresis / isoelectric focusing / electroblotting / Schiff's reagent