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Partially dephosphorylated α-casein with eight phosphorylation sites by AP was used as a sample. The sample was analyzed using the same 2-D procedures. Four isoelectric variants at Rf values of 0.22, 0.25, 0.30, and 0.35 in the IEF direction were observed in the 2-D separation coupling IEF-PAGE and normal SDS-PAGE. The calculated pI value of the fully dephosphorylated form (positioned at the Rf value of 0.22 in the IEF direction) was 4.8. In contrast to the general 2-D
procedure, the pI variants at the Rf values of 0.25 and 0.35 were separated into two spots on the 2-D gel coupling IEF-PAGE and Mn2+–Phos-tag SDS-PAGE (lower panel). Similarly, the 2-D procedure enabled the thick band at an Rf value of ca. 0.2 of the 1-D Mn2+–Phos-tag SDSPAGE
gel to separate as three spots at Rf values of 0.25, 0.30, and 0.35 in the IEF direction. These
results show that the separations of the protein phosphoisotypes are better by coupling IEF-PAGE and Mn2+–Phostag SDS-PAGE than by using the general 2-D procedure or 1-D Mn2+–Phos-tag SDS-PAGE. Faint bands (closed trianglethat are contaminants in the commercially available
α-casein were observed on the 1-D Mn2+–Phos-tag SDSPAGE gel.
Related data
α-casein