TWO-DIMENSIONAL ANALYSIS OF SKIM MILK PROTEINS USING PREPARATIVE ISOELECTRIC FOCUSING FOLLOWED BY POLYACRYLAMIDE GEL ELECTROPHORESIS

摘要

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 urea/1% triton X-100. Each of the 20 fractions obtained from the Rotofor device was then analyzed by polyacrylamide gel electrophoresis (PAGE). Both urea-PAGE and SDS-PAGE were used to separate purified caseins and skim milk resulting in comparable two-dimensional pattern. The major bovine caseins (αs1, αs2, β, κ-casein) were resolved better on urea-PAGE. The αs1- and β -casein were focused at pH ~ 4.5 and 4.8, respectively, whereas αs2-caseins focused as several bands at pH 6.2- 6.8. The A variant of κ -casein focuses 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 bans of all 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 of proteins in milk, as well as simplifies the detection of changes and foreign proteins in milk.