A thousand points of light: The application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics

Abstract

As proteomics evolves into a high-throughput technology for the study of global protein regulation, new demands are continually being placed upon protein visualization and quantitation methods. Chief among these are increased detection sensitivity, broad linear dynamic range and compatibility with modern methods of microchemical analyses. The limitations of conventional protein staining techniques are increasingly being encountered as high sensitivity electrophoresis methods are interfaced with automated gel stainers, image analysis workstations, robotic spot excision instruments, protein digestion work stations, and mass spectrometers. Three approaches to fluorescence detection of proteins in two-dimensional (2-D) gels are currently practiced: covalent derivatization of proteins with fluorophores, intercalation of fluorophores into the sodium dodecyl sulfate (SDS) micelle, and direct electrostatic interaction with proteins by a Coomassie Brilliant Blue-type mechanism. This review discusses problems encountered in the analysis of proteins visualized with conventional stains and addresses advances in fluorescence protein detection, including immunoblotting, as well as the use of charge-coupled device (CCD) camera-based and laser-scanner-based image acquisition devices in proteomics.