| Summary: | 碩士 === 國立成功大學 === 化學系碩博士班 === 91 === In this thesis, we investigated protein-protein interaction using microchip electrophoresis. In this design, the Cy5-labelled antigen was first incubated with the antibody to form the interaction complex in the free solution and then separated from the complex species by microchip electrophoresis. The evidence of forming the interaction complex was based on the detection of both the free and the complex species and the variation of the related the peak intensity by immuno-titration. The protein phosphorylation plays an important role in regulating cellular growth, differentiation, and migration, the experiment was designed to detect the protein phosphorylation using anti phosphorylation antibody as the affinity bite.
There are two major sections in the experiment. In the first section, we used the capillary electrophoresis to analyze a model system─anti-BSA and BSA and then analyze the target goal─phospho peptides and anti-phosphotyrosine. In the second section, we transferred the experiment from capillary electrophoresis to the microchip. In microchip electrophoresis, the sample injection and separation was integrated in a cross-microchannel which was etched on the glass substrate. The binding constant of the phospho peptide/phospho protein and the antibody was estimated by immuno-titration in which the concentration ratio between the antigen and the antibody was systematically varied. The result indicated that both the free form and complex form of comparing the FITC-peptide(pTyr) or Cy5-pTyr-BSA can be clearly detected and confirmed by the comparison with results obtained using a non-phosphorylated peptide or protein counterpart as the negative control. The binding constants for FITC-peptide(pTyr) or Cy5-pTyr-BSA with anti-phosphotyrosine, respectively was estimated to be 5.0×106 M-1 and 1.01×106 M-1.
For real sample analysis, we first investigated the matrix effect by spiking Cy5-pTyr-BSA into the cell lysate (transfection of the phoenix cell using two plasmid, pcDNA3.0-T7-Etk and pRL-c-SrcY527F) at various ratios. From the experiment, we found that the real sample may contain the strong BSA binding proteins. We further used FITC-anti-phosphotyrosine to react with cell lysate at different concentrations to detect the phospho-protein which was supposed to be present in the cell lysate. An obvious change of the peak shape for both the free and complex species was noticed. It was however difficult to differentiate the signal of the antibody and the complex, which requires further investigations.
Compared to the traditional immunoassay and capillary electrophoresis, the experiment time and the amount of the reagent can be greatly reduced using microchip electrophoresis w. Moreover, because of the shorter microchannel than capillary electrophoresis such that the interaction complex can be conserved and the analysis time was much shorter. Therefore, microchip electrophoresis is potentially capable of high-throughput screening of protein-protein interaction before the detection.
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