Comparative 2-DE protein analysis in a 3-D geometry gel.
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.
Ventzki
Robert
R
Scientific Core Facilities, Services & Technology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Rüggeberg
Sabrina
S
Leicht
Stefan
S
Franz
Thomas
T
Stegemann
Josef
J
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
D005782:Gels; D007202:Indicators and Reagents; D011506:Proteins