Two-dimensional Analysis of Skim Milk Proteins Using Preparative Isoelectric Focusing Followed by Polyacrylamide Gel Electrophoresis

A preparative isoelectric focusing (IEF) method was applied to separate skim milk proteins using the Rotofor device in a pH 3-10 gradient containing 4 M urealI % triton X-I 00. Each ofthe 20 fractions obtainedfrom the Rotofor device was then analyzed by polyacrylamide gel electrophoresis (PAGE). Both ureaPAGE and SDS-PAGE were used to separate purified caseins and skim milk resulting in comparable two-dimensional patterns. The major bovine caseins (ash lXsZ.I3. IC-casein) were resolved better on urea-PAGE. The lXsland l3-casein were focused at pH 4.5 and 4.8, respectively, whereas lXsz-caseinsfocused as several bands at pH 6.2-6.8. The A variant of IC-caseinfocuses at Fractions 6-9 which is slightly more acidic than the B variant that focuses at Fractions 7-13. No sample pretreatment was necessary to analyze skim milk proteins and urea-PAGE clearly resolved bands ofall major caseins and whey proteins. Preparative isoelectric focusing followed by PAGE was found to be a usefUl and powerful method to analyze milk proteins in two-dimensions. This technique facilitates the analysis of the relative amounts ofproteins in milk, as well as simplifies the detection of changes and foreign proteins in milk.