Biochemistry and molecular biology laboratory course（生物化学与分子生物学实验教程 英文版） azw3 chm 地址 kindle 阿里云 下载 umd pdf

《生物化学与分子生物学实验教程 Biochemistry and molecular biology laboratory course（英文版）》针对生物化学教学及研究中涉及的三类主要的生物分子——蛋白质、酶和核酸，根据生物大分子的理伦性质，结合实用的电泳技术、层次技术、离心技术、分子克隆技术等，综合系统地介绍生物大分子的分离、纯化、含量测定、分析鉴定和应用，并结合教学中物质代谢、酶促反应动力学的系统分析，设计实验并融入相关实验技术和研究新进展，使学生在实验教学中掌握知识、技能的同时，体会对科研方法的选择与评价。

目 录

Part 1 Basic techniques of biochemistry and molecularbiology

Chapter 1 Basic techniques of preparation of biomacromolecules 1

Section 1 Salting out 2

Section 2 Dialysis and ultrafiltration 3

Chapter 2 Spectrophotometry 5

Section 1 Basic principles 5

Section 2 The basic construction of spectrophotometers 7

Section 3 Some home-made spectrophotometers 9

Chapter 3 Chromatography 12

Section 1 Adsorption chromatography 12

Section 2 Partition chromatography 12

Section 3 Ion-exchange chromatography 14

Section 4 Gel filtration 17

Section 5 Affinity chromatography 18

Chapter 4 Electrophoretic techniques 20

Section 1 Basic principles 20

Section 2 Several types of commonly used electrophoresis 23

Chapter 5 Centrifugation techniques 29

Section 1 Basic principle 29

Section 2 General description of centrifugation 30

Chapter 6 Genetic engineering and analytical technology of gene expression 34

Section 1 Genetic engineering 34

Section 2 Polymerase chain reaction 40

Section 3 Nucleic acid hybridization 45

Section 4 DNA sequencing technology 46

Part 2 Experiments of the biochemistry and molecular biology

Chapter 7 Analysis of purification and physicochemical properties of proteins 49

Experiment 1 Colorimetric methods for protein determination 49

Experiment 2 Isolation and purification of serum γ-globulin 51

Experiment 3 Cellulose acetate membrane electrophoresis of serum proteins 53

Experiment 4 Determination of relative molecular mass of protein in SDS-PAGE 55

Chapter 8 Kinetics of enzyme-catalysed reactions 58

Experiment 1 The specificity of enzyme and effect of temperature, pH on the enzyme

Activity 58

Experiment 2 Isolation of alkaline phosphatase and determination of its specific activity 60

Experiment 3 Effects of substrates concentration of enzymatic activity: determination

of Km of alkaline phosphatase 64

Experiment 4 Inhibition effect of phosphate on alkaline phosphatase 66

Experiment 5 Analysis of lactic dehydrogenase isoenzyme 67

Chapter 9 Substance metabolism 70

Experiment 1 Extraction and identification of liver glycogen 70

Experiment 2 Determination of alanine transaminase activity (Reitman-Frankels’method) 71

Experiment 3 Enzyme catalyzed transamination of amino acids detected by paper

chromatography 73

Chapter 10 Basic experiment of molecular biology 76

Experiment 1 Preparation of the genomic DNA in the eukaryotic cells 76

Experiment 2 Determination of the concentration and purity of DNA by spectroscopy 77

Experiment 3 Agarose gel electrophoresis of nucleic acids 77

Experiment 4 Polymerase chain reaction (PCR) 79

Experiment 5 Isolation and purification of the DNA fragments 80

Experiment 6 DNA ligation—constructing recombinant plasmids 82

Experiment 7 Preparation of E.coli competent cells 83

Experiment 8 Transformation of Ca2+ competent cells 84

Experiment 9 The screening of recombinant DNA 85

Experiment 10 Minipreparation of plasmid DNA 86

Experiment 11 Digestion of plasmid DNA with restriction endonucleases 89

Experiment 12 Southern blot 90

Experiment 13 Expression of exogenous gene in prokaryotic cells 93

References 95

Appendix 96

Appendix Ⅰ The calculation table of ammonium sulfate saturation 96

Appendix Ⅱ Common buffer solution preparation 96

Appendix Ⅲ The relative molecular mass and pI of protein 99

Appendix Ⅳ Common data of chromatography 101

Appendix Ⅴ The conversion between centrifugation speed of centrifugation force 102

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Part 1 Basic techniques of biochemistry and molecularbiology

Chapter 1 B asic techniques of preparation of biomacromolecules

The process of preparation of biomacromolecules (BMM) includes selection of starting raw materials, disruption of cells and isolation of organelles, extraction, isolation and purification of BMM, and also concentration, drying and storage of them. The preparation of pure BMM is a tough and time-consuming task, which needs scrupulous care in every step to preserve the biological activity during the whole process. The selection methods used in the preparation of BMM depend upon the physical and chemical properties of BMM (Table 1-1) including size, shape, solubility, charges, etc. BMM with different structures and physical properties may be separated and purified by different methods.

Table 1-1 Methods of isolation and purification of BMM

Basis of separation Methods used

Molecular size and shape Differential centrifugation, ultrafiltration, molecular seive,dialysis

Solubility Salting out, extraction by solvents, partition chromatography,crystallization

Charges Electrophoresis, electroosmosis, isoelectric focusing electrophoresis,

ion exchange chromatography, adsorption chromatography

Specific biologic function Affinity chromatography

In order to assess the efficiency of each step in the process of isolation and purification, an assay method of the BMM intended to be isolated and purified should be established prior to the isolation process. With the assay method, the BMM can be traced in the separation process, and the purity and the yield (or recovery) can be calculated. The specific activity of the product obtained in each step should be calculated by dividing the total activity by the total amount of proteins present. Also the percentage recovery of the activity should also be calculated by dividing the total activity obtained in each step by the total activity of the first step, and then multiply by 100. Table 1-2 represents an example of such isolation and calculations, in which the enzyme is isocitrate dehydrogenase (ICD) from pig liver. From Table 1-2, we know that ICD has a 570-fold purification, and the yield is 25.5 %.

Table 1-2 Purification of isocitrate dehydrogenase from pig liver

In the following, brief discussions will be given to the basic techniques used in the process of isolation and purification of proteins, the most frequently encountered BMM.

Section 1 Salting out

This method is the earliest one used in the purification of proteins and enzymes. It is still used extensively now. The action of salt when in high concentration is to cause dehydration of the hydration layer of protein molecules, thus making the solubility of proteins decrease and, in turn, precipitate (salting out). Different proteins can be precipitated in different salt conc- entrations. On the contrary, the increase of solubility of proteins in low salt concentration is called “salting in”. During salting out, the relationship between solubility of proteins and the ionic strength (Chapter 4) of the solution can be expressed as follows:

Where S0 is the solubility of protein in pure water, I is the ionic strength of salt solution, S is the solubility of protein in solution with ionic strength I, Ks is the salting out constant. In the above equation, when temperature and pH keep constant, S0 is a parameter determined only by the nature of the protein. Therefore, under constant temperature and pH, S0 of the given protein is a constant. Let log S0=β

Then log S=β-Ks?I

The salting out constant Ks is primarily determined by the nature of the salt, e.g, valence and average radius of the ion, and is also determined by the nature of the protein. Different proteins have different Ks values in a given salt solution, the higher the Ks value is, the better salting out result will be obtained. From the above equation, one could see that different proteins will have different β and Ks values at a given temperature and pH environment. The method by which ionic strength is varied to achieve salting out of different proteins is called “Ks salt fractionation”. If the ionic strength is kept constant, the values of β might be changed by varying temperature and pH to achieve salting out of different proteins. This is called the method of “β salt fractionation”.

The following conditions should be considered when proteins are purified by using salting out method. 1. Salt species Commonly used in the salting out of proteins are neutral salts including ammonium sulfate, magnesium sulfate, sodium sulfate, sodium chloride and sodium phosphate. The most extensively used one is ammonium sulfate, which has the following advantages:

(1) High solubility. At 25 ℃, the solubility of ammonium sulfate could be more than 4.1mol/L (541 g/L). Each liter of water can solubilize about 767 g of ammonium sulfate. In such a wide range of salt concentrations, many proteins and enzymes could be precipitated by salting out method.

(2

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