Method optimization in two-dimensional gel electrophoresis

摘要

At present, two-dimensional gel electrophoresis (2-DE) is the method of choice for the separation of complex protein samples for subsequent proteome analysis. Although 2-DE is highly suited for the separation of complex protein samples it requires significant technical expertise to routinely produce qualitatively and quantitatively similar gels, partly due to the numerous experimental steps involved in the procedure. 2-DE consists of several key steps which include protein solubilization, Immoboline pH Gradient (IPG) strip rehydration/sample loading, IPG strip equilibration, and electrophoretic separation based upon the protein's isoelectric point and apparent molecular weight. Although the 2-DE approach has been standardized among different proteomic groups, many experimental factors still vary, some of which may significantly affect results. We observed several important factors that affect the 2-DE gel separation and protein yields. Here we present and discuss data and results involved in ouroptimization of 2-DE.