Electroblotting of proteins from polyacrylamide gels.

Abstract

Since the first complete genome sequence, that of the bacterium Haemophilus influenzae, was published in 1995 (1), a flurry of activity has seen the completion of the genomic sequences for more than 149 organisms (16 archael, 114 bacterial, and 19 eukaryotic). An up-to-date list of completed genomes is maintained on the GOLD website (Genomes OnLine Database, http://igweb.integratedgenomics.com/GOLD). Early in 2001, a major milestone was reached with the completion of the human genome sequence (2,3). A major challenge in the postgenome era will be to elucidate the biological function of the large number of novel gene products that have been revealed by the genome sequencing initiatives, to understand their role in health and disease, and to exploit this information to develop new diagnostic and therapeutic agents. The assignment of protein function will require detailed and direct analysis of the patterns of expression, interaction, localization, and structure of the proteins encoded by genomes; the area now known as “proteomics” (4). The ability of polyacrylamide gel electrophoresis (PAGE) techniques to resolve individual proteins of complex mixtures has resulted in this group of methods being indispensable to the protein biochemist. In particular, two-dimensional gel electrophoresis (2-DE) can routinely separate up to 2000 proteins from whole-cell and tissue homogenates, and the use of large format gels separations of up to 10,000 proteins have been described (5,6). For this reason, 2-DE remains the core technology of choice for protein separation in the majority of proteomics projects. Combined with the currently available panel of sensitive detection methods (7) and computer analysis tools (8), this methodology provides a powerful approach to the investigation of differential protein expression. Although gel electrophoresis procedures can provide characterization of proteins in terms of their charge (pI), size (Mr), relative hydrophobicity, and abundance, they give no direct clues as to their identities or functions. Fortunately, over the last years, a variety of sensitive methods have become available for the identification and charcaterization of proteins separated by gel electrophoresis. Conventional methods include reactivity with specific monoclonal and polyclonal antibodies, microsequencing by automated Edman degradation (9), and amino acid compositional analysis (10). More recently, techniques