One- and two-dimensional miniaturized electrophoresis of proteins with native fluorescence detection.
Miniaturized electrophoresis was successfully coupled with native fluorescence detection for direct analysis of proteins in one- and two-dimensional separations. The detection setup was based on direct observation of the UV-induced fluorescence of proteins using a CCD camera and a Hg (Xe) lamp for sample excitation. Protein mixtures were readily separated by size on a 1-cm segment of the one-dimensional gel in 8 min, and a detection limit of 0.04 ng per band was achieved. The dynamic range of the system was larger than 2 orders of magnitude. Miniaturized slab gel electrophoresis was performed on a special holder designed to couple isoelectric focusing with SDS-PAGE. Two-dimensional separation, including rehydration of IEF strip and fluorescence detection was completed in 2.5 h. Approximately 200 protein spots from Escherichia coli were detected on a 1 cm(2) area. A detection limit of 0.1 microg of total protein was achieved. The operation should be amenable to total automation.
Ames Laboratory-United States Department of Energy and Department of Chemistry, Iowa State University, Ames, Iowa 50011, USA.
Chanan
C
Sluszny
Edward S
ES
Yeung
D000818:Animals; D002417:Cattle; D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation* / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005453:Fluorescence; D007525:Isoelectric Focusing; D008904:Miniaturization / Q000295:instrumentation* / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*
D011506:Proteins