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Partially dephosphorylated β-casein by AP was analyzed. In the general 2-D separation
coupling IEF-PAGE and normal SDS-PAGE (upper panel), six isoelectric variants were observed at Rf values of 0.20, 0.27, 0.35, 0.38, 0.40, and 0.43 in the IEF direction. By collation with the separation in urea-PAGE, we assigned these isoelectric variants to the β-casein isotypes of 0P, 1P, 2P, 3P, 4P, and 5P, respectively.
The calculated pI value of the fully dephosphorylated form (Rf 0.20 in the IEF) was 5.1. The isoelectric variants located at the Rf 0.27 were detected as two spots. This result indicates that these phosphoisotypes have identical pI values but differ in their conformation in the presence of SDS. In contrast to the general 2-D procedure, the isoelectric variants at the Rf of
0.27, 0.35, and 0.38, in the IEF were separated as multiple spots on the 2-D gel coupling IEF-PAGE and Mn2+–Phos-tag SDS-PAGE (lower panel). The 2-D migration pattern was identical to that by 2-D separation coupling urea-PAGE and Mn2+–Phos-tag SDS-PAGE (urea-PAGE), and nine spots (1–9) were assigned, as shown. Each spot indicated by an arrow and an open triangle also corresponds to faint spots.
Related data
β-casein （Mn2+-Phos-tag）
β-casein（２D；Urea-PAGE／Mn2+-Phos-tag SDS-PAGE）