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内容简介
《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
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
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
摘要与插图
Part 1 Basic techniques of biochemistry and molecularbiologyChapter 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