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Development and Characterization of a 2-D microchip-based Protein separation system.

J.J. Feng, V Olazábal, J.A. Olivares, S. Sundaram, S. Krishnamoorthy and S. Iyer
Los Alamos National Laboratory, US

Keywords: 2DE, Simulation, Microchip, Capillary Electrophoresis

Traditional methods in proteomics have relied on gel-based separation of proteins followed by directed cleavage of these proteins and mass spectrometry to measure the products. The emphasis on reliable, high-throughput analyses has led to the development of novel pre-mass spec separation approaches such as multidimensional LC of typical peptides. While an effective technique, comparable expression levels cannot be determined using this approach unless some form of labeling is employed. Hence there is need for approaches that can suitably compare the effects of experimental treatment on intact proteins prior to peptide generation. The ideal separation system would allow real time imaging of the separation, use a labeling system that would enable a wide dynamic range of detection and facilitate interfacing with downstream procedures. To this end, we have developed a 2-D separation chip-based system (Figure 1) that employs capillary isoelectric focusing (CIEF) coupled with capillary zone electrophoresis (CZE) and uses a real-time fluorescent imaging of the proteins as they are separated. Design and development of such as system is not an easy task. One needs to understand the fundamental physico-chemical processes occurring in this system before making any design changes. In this regard, physics-based simulations can play a vital role. Here we present the results from one such study.

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