1. 金奈米粒子應用於偵測蛋白質間作用力與純化特定蛋白質 2. 螢光二氧化矽奈米粒子應用於鼠傷寒沙門氏桿菌的顯像
1. Detection of Protein-Protein Interactions and Purification of Specific Proteins with Substrate-Capped Gold Nanoparticles 2. Application of Fluorescent Silica Nanoparticles to image Salmonella typhimurium
Date Issued
2006
Date
2006
Author(s)
Tsai, Charng-Sheng
DOI
zh-TW
Abstract
Abstract
This thesis is divided into two parts. The first part used substrate-capped gold nanoparticles to detect protein-protein interactions and to purify specific protein. We designed and synthesized a series of sugar-capped gold nanoparticles (Sugar-GNPs) used in competition colorimetric assay for studying protein-protein interactions, particularly lectins. The competition assay uses the ensemble of lectin and Sugar-GNPs, which display blue color due to multivalent interactions between lectin and Sugar-GNPs, to identify the binding partners for lectin. Protein-Protein interaction can be evaluated qualitatively as well as quantitatively by using this competition assay. By using this methodology, a broad range of proteins can be rapidly evaluated for their abilities to interact with the protein of interest in real time, rendering high throughput screening possible.
Sugar-GNPs can be used not only in detection of protein-protein interaction but also in purification of carbohydrate-binding protein. If the surface plasmon resonance band is red-shifted, it indicates that the binding occurs between Sugar-GNPs and the protein in the solution. By centrifuging the colloid, the complex can be isolated and purified. The identity of the isolated carbohydrate-binding protein can be further confirmed by SDS-PAGE and MALDI-TOF-MS. The feasibility of Sugar-GNPs for the simultaneous enrichment and isolation of ConA from protein mixture is demonstrated. Furthermore, very few quantity of agglutinin can be purified from a small amount of dry weight of algae and the biological activity still remains after recovering the protein from the complex.
In addition, activity-based probe was designed and synthesized to form a covalent bond between the specific protein and substrate-capped gold nanoparticles through “click” reaction. Our experimental results indicate that labeled enzymes are easily separated from the unlabeled ones by centrifuge. SDS-PAGE and MALDI-TOF-MS are applied to analyze the purified samples.
In second part, two methods were employed to synthesize fluorescent silica nanoparticles, which were then modified by mannose derivatives on the surface, namely Man-F-SNP and Man-R-SNP. Both types of particles were used to stain Salmonella typhimurium for bacteria imaging and the results showed that photostability of Man-R-SNP is better than Man-F-SNP.
Subjects
金奈米粒子
螢光二氧化矽奈米粒子
Gold Nanoparticles
Fluorescent Silica Nanoparticles
Type
thesis
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