Ultrasensitive native fluorescence detection of proteins with miniaturized polyacrylamide gel electrophoresis by laser side-entry excitation

Direct detection of separated proteins inside polyacrylamide gels has many advantages compared to staining methods. Ultrasensitive native fluorescence detection of proteins with miniaturized 1-D and 2-D PAGE was achieved with laser side-entry excitation. The detection limit for R-phycoerythrin protein spots in 1-D SDS-PAGE with 532 nm excitation was as low as 15 fg, which corresponds to only 40,000 molecules. The average detection limit of six standard native proteins was 5 pg per band with 275 nm excitation. The dynamic range spanned more than three orders of magnitude. By using the same detection setup, approximately 150 protein spots from 30 ng of total Escherichia coli extraction were detected on a 0.8 cm x 1 cm gel in 2-D separation. The significant improvement in sensitivity for laser side-entry excitation comes from higher excitation power and lower background level compared with other excitation modes.