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.

The Collagen Binding Domain of Gelatinase A Modulates Degradation of Collagen IV by Gelatinase B

“…58 Unless otherwise noted, the various steps of the procedure were performed at room temperature and all incubation steps were performed under shaking conditions. After SDS-gel electrophoresis and Coomassie Blue staining, the protein bands of interest in the SDS gel were excised, cut into small pieces (1 × 1 mm), transferred into a 0.5-ml polyethylene sample vial and washed with 200 μl of nanopure water.…”

Electrotransfer of Immunoprobes through Thin-Layer Polyacrylamide Gels

“…We additionally observe that the electrical current decays over the 60 s of applied constant voltage in both of the electrotransfer configurations that included either the nanoporous membrane or no nanoporous membrane (Figure 4B). The decay in electrical current is attributed to products of electrolysis and Joule heating (SI Note 3), 22,34,35 and further study would build on the engineering design of similar electrotransfer systems to mitigate the impact of Joule heating and electrolysis on electrical current stability. 22,28,24 To next establish the intrinsic electrical conductivity of the nanoporous membrane, we calculated the electrical conductivity of the nanoporous membrane using the initial electrical current (t = 0 s).…”

BSPR/EBI 2007 meeting report – Integrative Proteomics: From Molecules to Systems July 25–27, 2007 Wellcome Trust Conference Centre, Hinxton, UK

“…Robert Ventzki (Heidelberg, Germany) gave an engaging talk on 3-D geometry gel electrophoresis. This intriguing but as yet relatively unused technique uses a 3-D gel block to separate up to 36 samples in parallel [6]. The technique itself seemed straightforward, however, it is questionable as to how much more reproducible and how much less variance there is in gels run in this manner in comparison to 2-D gels run using more conventional methodology.…”