Recent developments in two-dimensional gel electrophoresis with immobilized pH gradients: Wide pH gradients up to pH 12, longer separation distances and simplified procedures

Abstract

Wide-range immobilized pH 3-12 and 6-12 gradients were generated. Depending on the extraction method of sample preparation, proteins with p/s up to pH 11.7 were resolved. Highly reproducible protein patterns, focused to the steady-state with round-shaped spots up to the basic end were obtained. Moreover, because a strong water transport from cathode to anode (reverse electroendosmotic flow) inherent to narrow immobilized pH gradients (IPGs) exceeding pH 11, such as IPG 10-12, was negligible, the wide-range IPGs 3-12 and 6-12 could be run under standard conditions as originally described by Görg et al (Electrophoresis 1988, 9, 531-546). The wide-range immobilized pH gradient 3-12 proved to be perfectly suited for an overview separation of total cell extracts. Resolution could be increased by extending the separation distance from 18 to 24 cm. Furthermore, two-dimensional gel electrophoresis with IPGs (IPG-Dalt) was simplified by the use of an integrated system (IPGphor) where sample application by in-gel rehydration and isoelectric focusing (IEF) are performed automatically in a one-step procedure, overnight, without human assistance.