Isoelectric Focusing Electrophoresis

IEF separates proteins according to their isoelectric point (pi). A pH gradient in the gel is generally obtained using a mix of ampholytes and pharmalytes; samples are spotted on the surface of the gel and then an electric field is applied. When the protein reaches a zone of the gel with a pH equal to its pi, it loses its charges stopping its run. The IEF pattern of whole milk can be highly complex, since it can be affected by the phosphorylation forms and genetic variants. Mayer, Heidler, and Rockenbauer (1997) proposed the analysis of y2 and уз caseins by IEF in a pH range from 5 to 8 for a simultaneous evaluation of the presence of caprine and ovine milk in bovine milk. Similarly, Addeo, Moio, Chianese, and Nota (1989) suggest the use of the same caseins fractions to detect fraudulent addition of bovine milk to buffalo milk. Both works used the in vitro action of plasmin on milk or cheese to increase the sensibility of detection.

Another target polypeptide analyzed by IEF is the para к-casein, a polypeptide with a pi of 9.3, which shows a slight variability among bovine, sheep, and goat milk, as just discussed. Cheeses from the three species could be easily discriminated using IEF in the pH range 5-10 to analyze para к-casein (Figure et al., 1997).

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