Browsing A. James Clark School of Engineering by Subject "2-D PAGE"
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- ItemMultidimensional Microfluidic Bioseparation Systems With Spatial Multiplexing(2008-12-19) Yang, Shuang; DeVoe, Donald L; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Despite the refinement of liquid chromatography and peptide mass fingerprinting techniques for protein analysis, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) separations of intact proteins remain a core technology for proteomic studies due to their high peak capacities and resolving power. In 2-D PAGE, denatured intact proteins are separated on the basis of their charge state by isoelectric focusing (IEF), followed by a size-based separation using sodium dodecyl sulfate (SDS)-PAGE. While 2-D PAGE is most commonly practiced with backend analysis of proteins by mass spectrometry, 2-D PAGE expression maps alone can provide valuable insight for differential studies, including the analysis of post-translational modifications, by yielding information about the approximate isoelectric point (pI) and molecular weight (MW) of differentially expressed species within complex samples. However, conventional slab-gel 2-D PAGE remains a labor intensive and low throughput process, which significantly constrains its utility. In this dissertation, a novel microfluidic 2-D PAGE platform is developed which employs a combination of multifunctional photopolymerized polyacrylamide (PAAm) gels and a discontinuous sodium dodecyl sulfate (SDS)-PAGE buffer system. The PAAm gel is used as a highly-resolving separation medium for gel electrophoresis, while discrete PAAm gel plugs integrated into specific regions of the chip enable acid, base, and ampholyte solutions to be fully isolated prior to chip operation. The gel plugs allow different separation buffers to be stored within the chip, enabling the use of a discontinuous buffer system chosen to provide sample stacking during the second-dimension separation. The gel plugs are also employed as on-chip SDS containers, allowing defined volumes of SDS to be repeatably injected and complexed with the IEF-focused proteins, without the need for external intervention. The IEF channel itself possesses an angled geometry to minimize sample tailing, and the chip design employs backbiasing channels which eliminate sample leakage and enable uniform sample transfer between the separation dimensions. Validation of the full 2-D system is presented using fluorescently-labeled E. coli cell lysate as a model system.