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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Purification and characterization of glyceraldehyde 3-phosphate dehydrogenase from Chironomidae larvae. / 搖蚊幼蟲甘油醛3-磷酸脫氫酶之純化及分析 / Yao wen you chong gan you quan 3-lin suan tuo qing mei zhi chun hua ji fen xi

January 2010 (has links)
Chong, King Wai Isaac. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 99-104). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Table of Contents --- p.vi / Lists of Figures --- p.ix / List of Tables --- p.xi / List of Abbreviations --- p.xii / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- Overview of Glyceraldehyde 3-phosphate Dehydrogenases --- p.1 / Chapter 1.2 --- Properties And Molecular Structures of GAPDH --- p.3 / Chapter 1.3 --- Action Mechanism of GAPDH --- p.6 / Chapter 1.4 --- Novel Functions of GAPDH Unrelated to Carbohydrate Metabolism --- p.8 / Chapter 1.5 --- Effects of Heavy Metal on Enzyme Activity And Gene Expression of GAPDH --- p.10 / Chapter 1.6 --- Metal Binding Properties And Metal Binding Sites of GAPDH --- p.12 / Chapter 1.7 --- Isolation And Purification of GAPDH from Different Organisms --- p.13 / Chapter 1.8 --- Development of New Purification Method of GAPDH Using Immobilized Metal Affinity Chromatography --- p.15 / Chapter 1.9 --- Study of GAPDH from Chironomidae Larvae --- p.16 / Chapter 1.10 --- Aims of Study --- p.18 / Chapter Chapter Two: --- Methods And Materials --- p.19 / Chapter 2.1 --- Isolation of Native Chironomidae GAPDH --- p.19 / Chapter 2.1.1 --- Chemicals And Reagents --- p.19 / Chapter 2.1.2 --- Reagents --- p.19 / Chapter 2.1.3 --- Preparation of Crude Protein Extract from Chironomidae Larvae --- p.24 / Chapter 2.1.4 --- Immobilized Metal Affinity Chromatography --- p.24 / Chapter 2.1.5 --- Large Scale Preparation of Crude Protein Extract --- p.25 / Chapter 2.1.6 --- Ammonium Sulfate Fractionation --- p.25 / Chapter 2.1.7 --- Copper Affinity Column Chromatography --- p.26 / Chapter 2.1.8 --- Dye Affinity Column Chromatography --- p.26 / Chapter 2.2 --- Identification of Chironomidae GAPDH --- p.27 / Chapter 2.2.1 --- Chemicals And Reagents --- p.27 / Chapter 2.2.2 --- Reagents --- p.28 / Chapter 2.2.3 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.32 / Chapter 2.2.4 --- Non-Denaturing Polyacrylamide Gel Electrophoresis --- p.33 / Chapter 2.2.5 --- Protein Extraction from Coosmassie Blue Stained Polyacrylamide Gel --- p.33 / Chapter 2.2.6 --- N-terminal Amino Acid Analysis --- p.33 / Chapter 2.2.7 --- Sequence Analysis --- p.34 / Chapter 2.3 --- Kinetic Analysis of Chironomidae GAPDH --- p.34 / Chapter 2.3.1 --- Chemcials And Reagents --- p.34 / Chapter 2.3.2 --- Reagents --- p.34 / Chapter 2.3.3 --- Determination of Enzyme Concentration And GAPDH Activity --- p.35 / Chapter 2.4 --- Molecular Cloning of Chironomidae GAPDH --- p.36 / Chapter 2.4.1 --- Chemicals And Reagents --- p.36 / Chapter 2.4.2 --- Reagents --- p.37 / Chapter 2.4.3 --- RNA Extraction from Chironomidae Larvae --- p.41 / Chapter 2.4.4 --- DNase I Removal of Genomic DNA Contamination --- p.42 / Chapter 2.4.5 --- RNA Concentration Determination And RNA Agarose Electrophoresis --- p.42 / Chapter 2.4.6 --- First Strand cDNA Synthesis --- p.43 / Chapter 2.4.7 --- pRSet A B C Vectors --- p.43 / Chapter 2.4.8 --- Cloning Primer Design --- p.45 / Chapter 2.4.9 --- Polymerase Chain Reaction --- p.45 / Chapter 2.4.10 --- DNA Agarose Electrophoresis --- p.46 / Chapter 2.4.11 --- Restriction Enzyme Digestion of Insert And Plasmid --- p.46 / Chapter 2.4.12 --- Ligation of Plasmid And Insert DNA --- p.46 / Chapter 2.4.13 --- Preparation of Chemically Competent E. coli --- p.47 / Chapter 2.4.14 --- Transformation of Plasmid by Heat Shock --- p.47 / Chapter 2.4.15 --- Colony PCR --- p.48 / Chapter 2.5 --- Recombinant Protein Expression And Purification --- p.48 / Chapter 2.5.1 --- Chemicals And Reagents --- p.48 / Chapter 2.5.2 --- Reagents --- p.49 / Chapter 2.5.3 --- Protein expression by IPTG --- p.51 / Chapter 2.5.4 --- Protein purification by Nickel Affinity Column Chromatography --- p.52 / Chapter 2.5.5 --- EnterokinaseMax ´ёØ Removal of Polyhistidine Fusion Tag --- p.52 / Chapter 2.5.6 --- Western Blotting of Protein --- p.53 / Chapter Chapter Three: --- Results --- p.54 / Chapter 3.1 --- Two Affinity Chromatography Methods for GAPDH Purification --- p.54 / Chapter 3.2 --- Isolation And Purification of Native Chironomidae GAPDH --- p.54 / Chapter 3.3 --- Identification of Chironomidae GAPDH --- p.62 / Chapter 3.3.1 --- N-terminal amino acid analysis --- p.62 / Chapter 3.3.2 --- Sequence Analysis --- p.62 / Chapter 3.4 --- Molecular Cloning of Chironomidae GAPDH --- p.63 / Chapter 3.5 --- Isolation And Purification of recombinant Chironomidae GAPDH --- p.70 / Chapter 3.6 --- Protein Gel Electrophoresis Analysis of GAPDHs --- p.74 / Chapter 3.7 --- "Effects of Heavy Metals, pH And Temperature on GAPDHs" --- p.76 / Chapter 3.7.1 --- Heavy Metal Effect --- p.76 / Chapter 3.7.2 --- pH Effect --- p.76 / Chapter 3.7.3 --- Temperature --- p.77 / Chapter 3.8 --- Kinetic Analysis of GAPDHs --- p.84 / Chapter Chapter Four: --- Discussion --- p.89 / Chapter 4.1 --- New Method for The Isolation and Purification of Chironomidae GAPDH --- p.89 / Chapter 4.2 --- "Effects of Heavy Metals, pH And Temperature on GAPDHs" --- p.91 / Chapter 4.3 --- Kinetic Analysis of GAPDHs --- p.91 / Chapter 4.4 --- Zinc Activation of Chironomidae GAPDH --- p.92 / Chapter 4.5 --- Future Study --- p.93 / Chapter 4.5.1 --- Sequence Analysis Using Prediction Programmes --- p.94 / Chapter 4.5.2 --- Protein Crystallization --- p.95 / Chapter 4.5.3 --- Site-Directed Mutagenesis --- p.95 / Chapter 4.5.4 --- Biacore Surface Plasmon Resonance --- p.95 / Chapter Chapter Five: --- Conclusion --- p.98 / Chapter Chapter Six: --- References --- p.99

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