Abstract

Proteomics is a powerful technique to study proteomes extracted from biological sources. Proteome analysis classically is based on two-dimensional electrophoresis (2D-E) for protein separation and mass spectrometry (MS) for the protein identification. The serum protein analysis is a useful diagnosis that can be used as an indicator of the physiological or clinical status of a patient. One of the central and critical steps is sample application, therefore this method should be considered and optimized for 2-DE with immobilized pH gradient. The aim of this study was focused on comparative application of in-gel (rehydration loading) and in-cup (cup loading) sample application for 2-DE of human serum analysis. The results indicate that the number of spots detected with the rehydration loading washigher than the number obtained by cup-loading sample application. Also in this work, in order to obtain the best result with high resolution, a 2-DE procedure was optimized.

Key words: Proteomics, 2D-E, proteome, sample application.

Abbreviation

2-DE, Two-dimensional gel electrophoresis; MS, mass spectrometry; IPG, immobilized pH gradient; pI, isoelectric point; IEF, isoelectric focusing; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; DTT, dithiothreitol; TEMED, N,N,N',N' tetramethylethylenediamine.