Wednesday, 8 December 2004
13:00 - 13:30

Tools for Sample Preparation and Fractionation in 2D Electrophoresis

Anton Posch, Aran Paulus, Mary Grace Brubacher

Two-dimensional gel electrophoresis (2DE), with its unique capacity to resolve thousands of proteins in a single run, is a fundamental research tool for nearly all protein related scientific projects. Over the last two decades, numerous researchers in academia and in industry have improved the technology to the point where a novice user is capable of achieving respectable gel separations on the first try. In addition, 2DE technology as practiced today has seen enormous gains in reproducibility, resolution and automation all of which contribute to its widespread use. Nevertheless, 2DE is still a technically demanding method.
The quest to map and characterize each and every protein in a given cell type, tissue or organism has given 2DE an additional boost as the separation methodology of choice for many proteomics laboratories. However, the task list for a proteomics researcher is daunting: the number of proteins in a biological sample, although unknown at this time, is believed to be in the 100,000s, covering a concentration range of 7 or more orders of magnitude. In addition, the proteome is extremely dynamic, with protein expression depending on the cell state and further complicated by posttranslational modifications such as phosphorylation or glycolysation, to name just two possible changes to proteins in a functional biological system.
As more and more laboratories start up their own proteomic effort or ramp up existing programs, they realize that meticulous attention to 2DE methodology is only one critical aspect when identifying differentially expressed proteins or investigating a particular biological pathway.
The information content of 2DE is heavily influenced by a proper sample preparation strategy. Interestingly, not much attention was paid to this area during 2DE methodology development.
This talk is meant to provide a broad overview of the principles and recent developments of sample preparation tools prior to the first step of 2DE. Examples from three strategies for sample preparation, based on solution chemistry, chromatography and electrophoresis, will be discussed in detail as well as used to illustrate how these key areas can be applied to general-purpose sample cleanup and sample fractionation for enrichment of low abundance proteins.