CHED 927 |
| The goal of proteomics is to identify all of the proteins expressed in a cell. Conventional methods for proteome analysis generally are based on the use of two-dimensional (2-D) electrophoresis to identify cellular proteins. Protein patterns on 2-D gels are analyzed to generate proteome maps. Proteome maps of normal and diseased cells are compared to detect potential disease. The most abundant proteins can finally be identified using mass spectrometry (MS). However, conventional 2-D electrophoresis limits the use of proteomics for the identification of only markers of high abundance and the lower abundance proteins cannot be identified. The lack of sensitivity of conventional 2-D electrophoresis-based technology results primarily by a lack of resolving power because high abundance proteins mask the identification of low abundance proteins. Additionally, the polyacrylamide matrix typically used in conventional 2-D electrophoresis gives rise to a significant amount of background in the extracted sample. Microfluidic devices provide many applications for efficient proteomics analysis which only takes a few minutes. Our approach utilizes a polymeric 2-D microfluidic system for rapidly analyzing large numbers of cellular proteins involved in fetal calf serum (FCS) as a model obtained from the fetuses of cows. The FCS was fluorescently labeled with Alexa Fluor 633 dye, purified and thermally denatured before on-chip 2-D analysis was performed. For the 2-D separation, sodium dodecyl sulfate micro-capillary gel electrophoresis (SDS micro-CGE) was performed in the first dimension followed by micellar electrokinetic chromatography (MEKC) in the second dimension. The 2-D polymeric microchip channels were coated with methyl hydroxyl ethyl cellulose (MHEC) in order to suppress electroosmotic flow (EOF). Laser-induced fluorescence (LIF) was used for on-chip detection of conjugate FCS proteins. The average separation efficiency, resolution and peak capacity for separated proteins in generated 2-D electropherograms (protein landscapes) were >105 plates, 4 and 1000, respectively.
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Undergraduate Research Poster Session: Biochemistry
2:00 PM-4:00 PM, Monday, March 26, 2007 Hyatt Regency Chicago -- Riverside Center, Poster
Division of Chemical Education |