title: Comparative 2-DE protein analysis in a 3-D geometry gel.
abstract: Two-dimensional gel electrophoresis (2-DE) separation has not been considered suitable for large-scale comparative protein expression studies due to its limited throughput. We present a high-throughput analysis method based on three-dimensional (3-D) geometry gel electrophoresis. Following conventional isoelectric focusing (IEF), up to 36 immobilized pH gradient (IPG) strips are arrayed on the top surface of a 3-D gel body, and the samples transferred electrokinetically to the gel. A specific thermal management ensures that sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) occurs under identical electrophoretic and thermal conditions, avoiding gel-to-gel variations and thereby providing immediate comparability of the separation patterns. Proteins are Cy3-labeled for online detection of laser-induced fluorescence (LIF). Images are acquired by a digital camera and recorded as a 3-D image stack during electrophoresis. Image processing software decomposes the 3-D image stack into vertical sections representing conventional 2-DE slab gels, making results immediately accessible without further gel processing. The large number of simultaneously analyzed samples (n = 36) allows treating the sample index as a quasi-continuous experimental parameter (e.g., concentration, time, dose). The method offers a wide range of applications in molecular discovery, clinical diagnosis, pharmacology, and toxicology, like protein monitoring during disease development and screening of drug candidates for their effect on protein expression.
authors: Scientific Core Facilities, Services & Technology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors: Robert
authors: R
authors: Ventzki
authors: Sabrina
authors: S
authors: Rüggeberg
authors: Stefan
authors: S
authors: Leicht
authors: Thomas
authors: T
authors: Franz
authors: Josef
authors: J
authors: Stegemann
mesh_terms: D000818:Animals; D015195:Drug Design; D004563:Electrochemistry / Q000379:methods; D004586:Electrophoresis / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D005782:Gels; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007202:Indicators and Reagents / Q000494:pharmacology; D007525:Isoelectric Focusing / Q000379:methods*; D007700:Kinetics; D011506:Proteins / Q000032:analysis; D012984:Software; D013696:Temperature
chemical_list: D005782:Gels; D007202:Indicators and Reagents; D011506:Proteins