Automated protein analysis by online detection of laser-induced fluorescence in slab gels and 3-D geometry gels.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) still remains the most reliable and comprehensive analytical method for the evaluation of protein extracts. However, conventional SDS-PAGE is time-consuming and, thus, unpractical if several tens or hundreds of samples must be examined. We show that SDS-PAGE protein analysis can be automated using slab gel DNA sequencers and compare the instrument's performance with conventional SDS-PAGE in terms of resolution, sensitivity and sample capacity. Labeled protein bands are detected online by laser-induced fluorescence (LIF) and the acquired signals are electronically stored for further processing, avoiding gel staining and scanning. Appropriate software allows immediate display of recorded data and convenient evaluation. The method provides a higher sensitivity and dynamic range than conventional Coomassie-stained gels and the resolution of proteins with different masses is independent of the polyacrylamide concentration. Internal markers can also be used for direct quantification and assignment of the molecular masses. Additionally, we present a novel electrophoresis instrument for the simultaneous separation and online LIF detection of all samples of a microtiterplate in parallel lanes in a 3-D geometry gel cylinder. The specific gel thermostatting concept prevents irregular sample migration (smiling) and improves the reproducibility and comparability of individual separation patterns. In combination with the expected large capacity of 384 or 1,536 samples, this makes the instrument a valuable tool for high-throughput comparative screening applications.
Ventzki
Robert
R
Scientific Core Facilities, Services & Technology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Stegemann
Josef
J
Martinez
Lucia
L
de Marco
Ario
A
D001331:Automation / Q000379:methods; D002232:Carbocyanines / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D029968:Escherichia coli Proteins / Q000032:analysis; D005456:Fluorescent Dyes / Q000737:chemistry; D009862:Online Systems / Q000295:instrumentation*; D011506:Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity
D002232:Carbocyanines; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D011506:Proteins; C089187:cyanine dye 3