Automated ultra-thin-layer SDS gel electrophoresis of proteins using noncovalent fluorescent labeling.
Ultra-thin-layer SDS gel electrophoresis in conjunction with automated laser-induced fluorescence detection is a novel and powerful method for the analysis of fluorophore-labeled proteins. The technique described in this paper employs instant, noncovalent fluorophore labeling by the addition of a fluorescent 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 multilane format of SDS polyacrylamide slab gel electrophoresis and the high separation efficiency of capillary SDS gel electrophoresis, ultra-thin-layer SDS gel electrophoresis features rapid, high-throughput, and high-resolution analysis of proteins in the molecular mass range of 14-116 kDa. The good heat dissipation inherent to the ultrathin format enables the use of agarose and agarose-based composite separation matrixes, which can be easily replaced within the separation platform. Labeling efficiency as a function of the concentration of the staining dye, SDS, and proteins is thoroughly discussed. Detection sensitivity of the method was found to be at the low-femtomole 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.
Csapo
Z
Z
Genetic BioSystems, Inc., San Diego, California 92121, USA.
Gerstner
A
A
Sasvari-Szekely
M
M
Guttman
A
A
D001322:Autoanalysis; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D011506:Proteins