Automated ultra-thin-layer SDS gel electrophoresis of proteins using noncovalent fluorescent labeling

Ultra-thin-layer SDS gel electrophoresis in conjunction with automated lase r-induced fluorescence detection is a novel and powerful method for the ana lysis of fluorophore-labeled proteins. The technique described in this pape r employs instant, noncovalent fluorophore labeling by the addition of a fl uorescent staining dye to the sample proteins either during or immediately prior to the sample loading process. Thus, the method does not require time -consuming post- or preseparation staining/labeling. By combining the multi lane format of SDS polyacrylamide slab gel electrophoresis and the high sep aration efficiency of capillary SDS gel electrophoresis, ultra-thin-layer S DS gel electrophoresis features rapid, high-throughput, and high-resolution analysis of proteins in the molecular mass range of 14-116 kDa. The good h eat dissipation inherent to the ultrathin format enables the use of agarose and agarose-based composite separation matrixes, which can be easily repla ced within the separation platform. Labeling efficiency as a function of th e concentration of the staining dye, SDS, and proteins is thoroughly discus sed. Detection sensitivity of the method was found to be at the low-femtomo le level (1.25 ng/band), determined by analyzing a set of serial dilutions of standard proteins. Practical example of molecular mass determination and characterization of a complex protein mixture are also shown.