An integrated strategy combining in-solution isoelectric focusing (OFFGEL) and differential in-gel electrophoresis (DIGE): Visualization of A1 and A2 variants of cow´s milk β-casein

Two dimensional (2D) – differential in-gel electrophoresis (DIGE) represented a significant advancement in protein separation, enabling multiplexed analysis of fluorescently labeled proteins within a single gel. Despite its analytical strengths, its widespread use was limited by the cost of fluorescent dyes and the requirement for specialized laser scanners. In this study, we present a strategy that combines DIGE with in-solution isoelectric focusing (OFFGEL). Recovering proteins in-solution allows greater flexibility in protein loading for the first dimension, since resulting fractions can be multiplexed for additional analyses beyond second-dimension separation. Gels were scanned using a charge-coupled device (CCD) based cost-effective imaging system. The usefulness of this approach was demonstrated by visualizing A1 and A2 variants of β-casein (β-CN) in cow milk, which differ by a single amino acid. Milk samples were labeled, fractionated by OFFGEL, and fractions further resolved using SDS-PAGE. The differential migration of A1 and A2 β-CN variants according to their isoelectric point (pI) was visualized and quantified. Results showed protein band patterns consistent with theoretical pI differences between variants. LC-MS/MS analysis confirmed the presence of A1, A2, or both. Results demonstrated the potential of integrating OFFGEL and DIGE as a robust and affordable strategy for comparative proteomics of protein subsets.

Instituto de Agroquímica y Tecnología de Alimentos
Leartiker