The first edition of this manual presents shortened versions of the basic biochemistry methods designed for use at the lab bench in a biochemistry and molecular biology laboratory. It is primarily intended for undergraduate medical students, and hopefully it will be a useful reference book for graduate students and teaching staff. The objectives for the course are for students to learn modern biochemical laboratory methodologies and techniques, develop the ability to perform experiments successfully and independently, properly interpret the data and write scientific laboratory reports. Students who learn biochemistry as a part of medical science should be able to understand the concepts and methods of not only the conventional clinical laboratory tests but also the recently developed tests. For this purpose, we have made this manual with incorporation of some clinical laboratory test kits. All of the experiments have been performed many times in our own laboratory. We hope that they will provide a reliable training of the most important and commonly used techniques of contemporary biochemistry.
We express our appreciation to our colleagues in the Department of Biochemistry and Molecular Biology, Wuhan University School of Medicine, who have contributed ideas, techniques and experiences to the manual. We are really grateful to all the experts, especially those from other universities, who made big contribution to the compiling of various chapters.
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目录
Chapter Ⅰ Spectrophotometry
1.1 Basic Concepts and Principle of Spectrophotometry
1.2 Measurement..of Light Absorption
1.3 Aplications of Spectrophotometry
EXP.1 Quantitative Analysis of Protein
EXP.2 Determination of Blood Glucose
Chapter Ⅱ Electrophoresis
2.1 Basic Principles of EIectr0Dhoresis
2.2 lmpact Factors of EIectrophoresis
2.3 Detection of Components after Electrophoretic Separation
2.4 Special Electrophoretic Techniques and Applications
EXP.3 Separation of Serum Proteins by Cellulose Acetate Membrane Electrophoresis
EXP.4 Separation of Serum Lipoproteins by Agarose Gel Electrophoresis
EXP.5 Proteins Separation by SDS·Polyacrylamide Gel Electrophoresis
Chapter Ⅲ Chromatography
3.1 Fundamental Principles of Chromatography
3.2 Commonly Used Chromatographic Techniques
EXP.6 Detecting Transamination of Amino Acids by Paper Chromatography
EXP.7 Separation of Mixed Amino Acids by Cation Exchange Chromatography
EXP.8 Separation of Hemoglobin and Dinitrophenyl（DNP）一glutamate by Gel Filtration Chromatography
Chapter Ⅳ Enzyme Analysis
4.1 The Activity of Enzyme
4.2 Enzyme kinetics（Factors affecting reaction velocity）
4.3 Enzymatic analysis
EXP.9 Assay the Activity of Alanine Aminotransferase（ALT）in Serum（Mohun’S Method）
EXP.10 Lactate Dehydrogenase（LDH）Analysis
EXP.1 1 Effect of Substrate Concentration on Enzyme Activity-Determining Km Value for Alkaline Phosphatase
EXP.1 2 Coupled enzymatic reaction assay（EnzymaticEndpoint Method）- Determination of Total Cholesterol，Triglycerides and Glucose in Serum
Chapter Ⅴ solation and Purification of Protein
5.1 Methods and Basic Principles of Protein Purification
5.2 Procedure of Protein Preparation
5.3 Crystallization，Concentration，Drying and Storage of Protein
5.4 ldentification and Analysis of Protein
EXP.13 Isolation and Identification of Serum IgG
EXP.14 Expression.Puriftcation and Identification of Glutathione S-Transferase Fusion Protein
Chapter Ⅵ solation，Purification and Identification of Nucleic Acids
6.1 lsolation and Purification of Nucleic Acids
6.2 Identiflcation and Analysis of Nucleic Acids
EXP.1 5 Isolation of Eukaryotic Genomic DNA by Proteinase K.Phenol or NaI Method
EXP.16 SDS.Alkaline Lysis of Plasmid DNA
EXP.17 Isolation of Total RNA bv TRIzol Reagent
EXP.18 Identification of DNA by UV-Spectrophotometry and Gel Eleetrophoresis
EXP.19 Identification of RNA by UV-Spectmphotometry and Formaldehyde Denaturating Agarose Gel Electrophoresis
EXP.20 Polymerase Chain Reaction（PCR）and Reverse Transcription-PCR
Chapte Ⅶ Genetic Engineering
7.1 The Process of Gene Cloning
7.2 Enzyrues for Gene Cloning
7.3 Vectors for Gene Cloning
7.4 Expression of Fo reign Genes and Purification of Recombinant Proteins
EXP.21 DNA Cloning
EXP.22 Expression of Exogenous Gene in E.coli and Purification of the Expressional Product
English-Chinese Index
The first edition of this manual presents shortened versions of the basic biochemistry methods designed for use at the lab bench in a biochemistry and molecular biology laboratory. It is primarily intended for undergraduate medical students, and hopefully it will be a useful reference book for graduate students and teaching staff. The objectives for the course are for students to learn modern biochemical laboratory methodologies and techniques, develop the ability to perform experiments successfully and independently, properly interpret the data and write scientific laboratory reports. Students who learn biochemistry as a part of medical science should be able to understand the concepts and methods of not only the conventional clinical laboratory tests but also the recently developed tests. For this purpose, we have made this manual with incorporation of some clinical laboratory test kits. All of the experiments have been performed many times in our own laboratory. We hope that they will provide a reliable training of the most important and commonly used techniques of contemporary biochemistry.
We express our appreciation to our colleagues in the Department of Biochemistry and Molecular Biology, Wuhan University School of Medicine, who have contributed ideas, techniques and experiences to the manual. We are really grateful to all the experts, especially those from other universities, who made big contribution to the compiling of various chapters.
--------------------------------------------------------------------------------
目录
Chapter Ⅰ Spectrophotometry
1.1 Basic Concepts and Principle of Spectrophotometry
1.2 Measurement..of Light Absorption
1.3 Aplications of Spectrophotometry
EXP.1 Quantitative Analysis of Protein
EXP.2 Determination of Blood Glucose
Chapter Ⅱ Electrophoresis
2.1 Basic Principles of EIectr0Dhoresis
2.2 lmpact Factors of EIectrophoresis
2.3 Detection of Components after Electrophoretic Separation
2.4 Special Electrophoretic Techniques and Applications
EXP.3 Separation of Serum Proteins by Cellulose Acetate Membrane Electrophoresis
EXP.4 Separation of Serum Lipoproteins by Agarose Gel Electrophoresis
EXP.5 Proteins Separation by SDS·Polyacrylamide Gel Electrophoresis
Chapter Ⅲ Chromatography
3.1 Fundamental Principles of Chromatography
3.2 Commonly Used Chromatographic Techniques
EXP.6 Detecting Transamination of Amino Acids by Paper Chromatography
EXP.7 Separation of Mixed Amino Acids by Cation Exchange Chromatography
EXP.8 Separation of Hemoglobin and Dinitrophenyl（DNP）一glutamate by Gel Filtration Chromatography
Chapter Ⅳ Enzyme Analysis
4.1 The Activity of Enzyme
4.2 Enzyme kinetics（Factors affecting reaction velocity）
4.3 Enzymatic analysis
EXP.9 Assay the Activity of Alanine Aminotransferase（ALT）in Serum（Mohun’S Method）
EXP.10 Lactate Dehydrogenase（LDH）Analysis
EXP.1 1 Effect of Substrate Concentration on Enzyme Activity-Determining Km Value for Alkaline Phosphatase
EXP.1 2 Coupled enzymatic reaction assay（EnzymaticEndpoint Method）- Determination of Total Cholesterol，Triglycerides and Glucose in Serum
Chapter Ⅴ solation and Purification of Protein
5.1 Methods and Basic Principles of Protein Purification
5.2 Procedure of Protein Preparation
5.3 Crystallization，Concentration，Drying and Storage of Protein
5.4 ldentification and Analysis of Protein
EXP.13 Isolation and Identification of Serum IgG
EXP.14 Expression.Puriftcation and Identification of Glutathione S-Transferase Fusion Protein
Chapter Ⅵ solation，Purification and Identification of Nucleic Acids
6.1 lsolation and Purification of Nucleic Acids
6.2 Identiflcation and Analysis of Nucleic Acids
EXP.1 5 Isolation of Eukaryotic Genomic DNA by Proteinase K.Phenol or NaI Method
EXP.16 SDS.Alkaline Lysis of Plasmid DNA
EXP.17 Isolation of Total RNA bv TRIzol Reagent
EXP.18 Identification of DNA by UV-Spectrophotometry and Gel Eleetrophoresis
EXP.19 Identification of RNA by UV-Spectmphotometry and Formaldehyde Denaturating Agarose Gel Electrophoresis
EXP.20 Polymerase Chain Reaction（PCR）and Reverse Transcription-PCR
Chapte Ⅶ Genetic Engineering
7.1 The Process of Gene Cloning
7.2 Enzyrues for Gene Cloning
7.3 Vectors for Gene Cloning
7.4 Expression of Fo reign Genes and Purification of Recombinant Proteins
EXP.21 DNA Cloning
EXP.22 Expression of Exogenous Gene in E.coli and Purification of the Expressional Product
English-Chinese Index
