Title

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

Author Info

Hye-Hyun Y. Kim, University of Illinois at Urbana-Champaign
Rafael Jiménez-Flores, University of Illinois at Urbana-ChampaignFollow

Publication Date

10-1992

Recommended Citation

Published in Journal of Food Biochemistry, Volume 16, Issue 5, October 1, 1992, pages 307-321.

NOTE: At the time of publication, the author Rafael Jiménez-Flores was not yet affiliated with Cal Poly.

The definitive version is available at https://doi.org/10.1111/j.1745-4514.1992.tb00454.x.

Abstract

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 (α_s1, α_s2, β, κ-casein) were resolved better on urea-PAGE. The α_s1- and β -casein were focused at pH ~ 4.5 and 4.8, respectively, whereas α_s2-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.

Disciplines

Dairy Science

Copyright

1993 Wiley Periodicals, Inc.