Chapter 7 Separation of protein

实验四蛋白质印迹分析【实验目的】了解蛋白质印迹法的基本原理及其操作和应用。

【实验原理】蛋白质印迹法又称为免疫印迹法，这是一种可以检测固定在固相载体上蛋白质的免疫化学技术方法。

待测蛋白既可以是粗提物也可以经过一定的分离和纯化, 另外这项技术的应用需要利用待测蛋白的单克隆或多克隆抗体进行识别。

如图所示，可溶性抗原，也就是待测蛋白首先要根据其性质，如分子量，分子大小，电荷以及其等电点等采用不同的电泳方法进行分离；通过电流将凝胶中的蛋白质转移到聚偏二氟乙烯膜上；利用抗体(一抗) 与抗原发生特异性结合的原理，以抗体作为探针钓取目的蛋白。

值得注意的是在加入一抗前应首先加入非特异性蛋白，如牛血清白蛋白对膜进行“封阻” 而防止抗体与膜的非特异性结合。

经电泳分离后的蛋白往往需再利用电泳方法将蛋白质转移到固相载体上, 我们把这个过程称为电泳印迹。

常用的两种电转移方法分别为:1.半干法: 凝胶和固相载体被夹在用缓冲溶液浸湿的滤纸之间,通电时间为10 分钟~30 分钟。

2. 湿法：凝胶和固相载体夹心浸放在转移缓冲溶液中，转移时间可从45 分钟延长到过夜进行。

由于湿法的使用弹性更大并且没有明显浪费更多的时间和原料，因此我们在这里只描述湿法的基本操作过程。

对于目的蛋白的识别需要采用能够识别一抗的第二抗体。

该抗体往往是购买的成品，已经被结合或标记了特定的试剂，如辣根过氧化物酶。

这种标记是利用辣根过氧化物酶所催化的一个比色反应，该反应的产物有特定的颜色且固定在固相载体上，容易鉴别。

因此可通过对二抗的识别而识别一抗，进而判断出目标蛋白所在的位置。

其他的识别系统包括碱性磷酸酶系统和125I 标记系统。

【实验材料】1. 实验器材SDS/PAGE实验相关材料；电转移装置；供电设备；PVDF膜 ( Millipore Immobion-P #IPVH 000 10 ); Whatman 3MM纸；其他工具：镊子、海绵垫、剪子、手套、小塑料或玻璃容器、浅盘。

蛋白组学英语English:Proteomics, also known as proteinomics, is the study of the entire complement of proteins present in a cell, tissue, or organism. This field aims to understand the structure, function, and interactions of proteins on a large scale. It involves the identification, quantification, and characterization of proteins, as well as the study of their post-translational modifications. Proteomics techniques include protein separation methods, mass spectrometry, and bioinformatics analysis. The applications of proteomics are wide-ranging, including biomarker discovery, drug development, and understanding disease mechanisms. Overall, proteomics plays a crucial role in advancing our understanding of the complex biological processes that occur at the molecular level.中文翻译:蛋白组学，也称为蛋白质组学，是研究细胞、组织或生物体中存在的全部蛋白质的领域。

2021 年 2 月 Journal of Chemical Engineering of Chinese Universities Feb. 2021文章编号：1003-9015(2021)01-0001-12连续流层析及用于抗体分离的新进展荆淑莹, 史策, 姚善泾, 林东强(浙江大学生物质化工教育部重点实验室, 浙江大学化学工程与生物工程学院, 浙江杭州 310027)摘要：连续生物制造是生物制药的发展趋势，其中连续流层析是关键环节。

作者根据近年来国内外连续流层析的研究进展，着重介绍了产物捕获和精制阶段的连续流层析技术，分析了不同模式的技术差异、各自特点和应用现状。

针对未来发展趋势，进一步介绍了整合连续流层析过程，以及用于抗体连续生产的难点和挑战。

作为一项新兴技术，抗体连续生产具有提高过程产率和产品质量、促进设备小型化和流程自动化、拓展过程灵活性和可靠性、降低生产成本等显著优势，但尚有不少方面需要改进和深化，包括过程设计、过程分析技术和过程控制技术等，特别是基于模型的预测分析和控制方法。

关键词：连续流层析；抗体；捕获；精制；过程集成；过程分析技术中图分类号：TQ028.8 文献标志码：A DOI：10.3969/j.issn.1003-9015.2021.01.001 Progress on continuous chromatography and its application in antibody separationJING Shu-ying, SHI Ce, YAO Shan-jing, LIN Dong-qiang(Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China)Abstract: Continuous chromatography is a key unit of continuous biomanufacturing which is the trend in biopharmaceutical industry. The continuous chromatographic technologies for protein capture and polishing were reviewed based on recent research progress. Technical differences, characteristics and current applications of different separation modes are focused. Integrated continuous chromatography is introduced and the challenges for continuous production of antibodies are discussed considering future development. Continuous manufacturing has the potentials to increase productivity and product quality, reduce footprint and costs, and enhance process automatization, flexibility and reliability. However, more studies such as process design, process analytical and control technologies, are necessary to improve continuous manufacturing processes, especially for model-based predictive analysis and control strategies.Key words: continuous chromatography; antibody; capture; polishing; process integration; process analytical technology1引言单克隆抗体(简称单抗)药物具有靶向性强、疗效好、副作用小等特点，在治疗癌症、自身免疫性疾病等方面具有显著优势[1-2]。

【实验目的】了解蛋白质印迹法的基本原理及其操作和应用。

【实验原理】蛋白质印迹法又称为免疫印迹法，这是一种可以检测固定在固相载体上蛋白质的免疫化学技术方法。

待测蛋白既可以是粗提物也可以经过一定的分离和纯化,另外这项技术的应用需要利用待测蛋白的单克隆或多克隆抗体进行识别。

如图所示，可溶性抗原，也就是待测蛋白首先要根据其性质，如分子量，分子大小，电荷以及其等电点等采用不同的电泳方法进行分离；通过电流将凝胶中的蛋白质转移到聚偏二氟乙烯膜上；利用抗体（一抗）与抗原发生特异性结合的原理，以抗体作为探针钓取目的蛋白。

值得注意的是在加入一抗前应首先加入非特异性蛋白，如牛血清白蛋白对膜进行“封阻”而防止抗体与膜的非特异性结合。

经电泳分离后的蛋白往往需再利用电泳方法将蛋白质转移到固相载体上,我们把这个过程称为电泳印迹。

常用的两种电转移方法分别为: 1.半干法: 凝胶和固相载体被夹在用缓冲溶液浸湿的滤纸之间,通电时间为10分钟~30分钟。

2.湿法：凝胶和固相载体夹心浸放在转移缓冲溶液中，转移时间可从45分钟延长到过夜进行。

由于湿法的使用弹性更大并且没有明显浪费更多的时间和原料，因此我们在这里只描述湿法的基本操作过程。

对于目的蛋白的识别需要采用能够识别一抗的第二抗体。

该抗体往往是购买的成品，已经被结合或标记了特定的试剂，如辣根过氧化物酶。

这种标记是利用辣根过氧化物酶所催化的一个比色反应，该反应的产物有特定的颜色且固定在固相载体上，容易鉴别。

因此可通过对二抗的识别而识别一抗，进而判断出目标蛋白所在的位置。

其他的识别系统包括碱性磷酸酶系统和125I标记系统。

【实验材料】1. 实验器材 SDS/PAGE实验相关材料；电转移装置；供电设备；PVDF膜（Millipore Immobion-P #IPVH 000 10）；Whatman 3MM 纸；其他工具：镊子、海绵垫、剪子、手套、小塑料或玻璃容器、浅盘。

2. 实验试剂⑴ 10x转移缓冲溶液（1L）：30.3g Trizma base(0.25M), 144 g甘氨酸(1.92M),加蒸馏水至1L, 此时pH约为8.3，不必调整。

Protein A/G MagBeads Cat. No. L00277Technical Manual No. TM0249 Version 08212013 Index1.Product Description2. Instruction For Use3.Troubleshooting4. General Information1.Product Description1.1Intended UseGenScript Protein A/G MagBeads are ideal for small‐scale antibody purification and immunoprecipitation (IP) of proteins, protein complexes or other antigens.1.2PrincipleThe sample containing antibody is added to the Protein A/G MagBeads. The antibody will bind to beads during a short incubation. Then the beads‐bound antibody may be eluted from the beads by using a magnetic separation rack, or used for immunoprecipitation (IP). A cross‐linking procedure may be needed before IP to prevent co‐elution of the primary antibody. Magnetic separation eliminates the changes of micro tubes, minimizes the loss of sample and removes excessive steps of traditional centrifugation method.1.3Description of MaterialMaterial SuppliedGenScript Protein A/G MagBeads are super paramagnetic beads of average 40 μm in diameter, covalently coated with recombinant Protein A/G. The beads are supplied as 25% slurry in phosphate buffered saline (PBS), pH 7.4, containing 20% ethanol. The Protein A/G MagBeads have a binding capacity of more than 10 mg Goat IgG per 1 ml settled beads (e.g. 4 ml 25% slurry).Protein A/G is a genetically engineered protein (MW≈43 kDa) that combines the IgG binding sites of both Protein A and Protein G. 6×His‐tag was attached to its N‐terminal to facilitate the purification. The secreted Protein A/G contains four Fc‐binding domains from Protein A and two from Protein G, making it a more universal tool to bind and purify immunoglobulins.Cat. No. L00277 Size: 2 ml.Additional Material RequiredMixing/Rotation DeviceMagnetic Separation RackTest tubes and pipettesBuffers and solutions (see below)Additional Buffers NeededBinding/Wash Buffer: 20 mM Na2HPO4, 0.15 M NaCl, pH 7.0Elution Buffer: 0.1 M glycine, pH 2‐3Neutralization Buffer: 1 M Tris, pH 8.51×SDS Sample Buffer: 62.5 mM Tris‐HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT,0.01% w/v bromophenol blue2.Instruction For UseThe protocol uses 100 μl Protein A/G MagBeads, this may be scaled up or down accordingly.2.1Preparation of the MagBeadspletely resuspend the beads by shaking or vortexing the vial.2.Transfer 100 μl beads into a clean tube.3.Place the tube on a magnetic separation rack to collect the beads. Remove and discard the supernatant.4.Add 1 ml Binding/Wash Buffer to the tube and invert the tube several times to mix. Use the magnetic separationrack to collect the beads and discard the supernatant. Repeat this step twice.2.2Separation of Target IgG1.Resuspend the beads in 100 μl Binding/Wash Buffer.2.Add the sample containing target IgG to the tube and gently invert the tube to mix.3.Incubate the tube at room temperature with mixing (on a shaker or rotator) for 30 – 60 minutes.e the magnetic separation rack to collect the beads and discard the supernatant. If necessary, keep thesupernatant for analysis.5.Add 1 ml Binding/Wash Buffer to the tube and mix well, use the magnetic separation rack to collect the beads anddiscard the supernatant. Repeat the wash step three more times.6.Proceed to elution of isolated IgG (Section 2.3).2.3Elution of Isolated IgG1.Add 100 μl Elution Buffer to the tube and mix well. Incubate for five minutes at room temperature with occasionalmixing.e the magnetic separation rack to collect the beads and transfer the supernatant that contains the eluted IgG intoa clean tube.3.Repeat Step 1 and 2 twice.4.Add 10 μl of Neutralization Buffer to each 100 μl eluate to neutralize the pH. If needed, perform a buffer exchangeby dialysis or desalting.2.4ImmunoprecipitationBound IgG will be co‐eluted along with the target when using elution methods. If the presence of IgG does not disturb desired detection system, go directly to section 2.4.2 below. For applications where co‐elution of the IgG is not desired, the primary IgG can be cross‐linked to the Protein A/G MagBeads as described in section 2.4.1 below.2.4.1Cross‐linking IgG to the Beads1.Add 1 ml 0.2 M triethanolamine, pH 8.2 to the Protein A/G MagBeads with immobilised IgG. Wash twice using themagnetic separation rack with 0.2 M triethanolamine, pH 8.2 as the washing buffer.2.Resuspend the beads in 1 ml of 20 mM dimetyl pimelimidate dihydrochloride (DMP) in 0.2 M triethanolamine, pH 8.2(5.4 mg DMP/ml buffer). This cross‐linking solution must be prepared freshly.3.Incubate the beads with rotational mixing for 30 minutes at room temperature. Use the magnetic separation rack tocollect the beads and discard the supernatant.4.Resuspend the beads in 1 ml of 50 mM Tris, pH 7.5 to stop the reaction and incubate for 15 minutes at roomtemperature with rotational mixing.e the magnetic separation rack to collect the beads and discard the supernatant.6.Wash the cross‐linked beads three times with 1 ml PBS, pH7.4.2.4.2Binding Antigen to the IgG Cross‐linked Beads1.Add sample containing target antigen to the beads. For a 100 kD protein, use a volume containing approximate 25 µgtarget antigen/ml beads to assure an excess of antigen. If dilution of antigen is necessary, PBS or 0.1 M phosphate buffer (pH 7‐8) can be used as dilution buffer.2.Incubate with tilting and rotation for one hour at room temperature.3.Place the tube on the magnetic separation rack for 2 minutes to collect the IgG‐coated Beads‐target complex. Forviscous samples, double the time on the rack. Discard the supernatant.4.Wash the beads 3 times using 1 ml PBS.2.4.3Elution of Target ProteinA.Denaturing elution1.Place the tube from section2.4.2 on the magnetic separation rack to collect the beads and discard the supernatant.2.Add 100 µl 1XSDS Sample Buffer to the tube and mix well.3.Heat the tube at 100°C for five minutes.e the magnetic separation rack to collect the beads and transfer the supernatant containing desired sample into anew tube.5.Analyze the sample by SDS‐PAGE followed by Western blot analysis.B.Non‐denaturing elution1.Place the tube from section2.4.2 on the magnetic separation rack to collect the beads and discard the supernatant.2.Add 100 µl Elution Buffer to the tube and mix well. Incubate for five minutes at room temperature with occasionalmixing.e the magnetic separation rack to collect the beads and transfer the supernatant into a new tube.4.Repeat Step 2 and 3 twice.5.Add 10 μl Neutralization Buffer to each 100 μl of eluate to neutralize the pH.3.TroubleshootingReview the information below to troubleshoot your experiments using the GenScript Protein A/G MagBeads. Problem Possible Cause SolutionThe beads are difficult toimmobilize using the magneticseparation rack.Too many beads are used.Decrease the volume of MagBeadssuspension.A considerable amount of samplehas been added, but very fewspecific antibody of interest isdetected.The antibody of interest is at verylow concentration.Use a serum‐free medium for cellsupernatant samples.Affinity‐purify the antibody using itsspecific antigen coupled to anaffinity supporting material.The antibody of interest is purified,but it is degraded (as determined byloss of function in downstreamassay).The antibody is sensitive to low‐pHelution buffer.The downstream application issensitive to the neutralized elutionbuffer.Try another elution reagent, such as3.5 M MgCl2, 10 mM phosphate, pH7.2.Desalt or dialyze the eluted sampleinto a suitable buffer.No antibody is detected in anyeluate.The antibody in the sample cannotbind to Protein A/G.Try GenScript Protein A MagBeadsor Protein G MagBeads.4.General Information4.1Storage and StabilityThis product is stable until the expiration date stated on the COA, when stored unopened at 2–8°C. Do not freeze the product. Keep the MagBeads in liquid suspension during storage and all handling steps. Drying will cause loss of binding capacity and result in reduced performance. Resuspend the beads well before use. Be careful to avoid bacterial/fungal contamination.4.2Technical SupportPlease contact GenScript for further technical information (see contact details). Certificate of Analysis/Compliance is available upon request. The latest revision of the package insert/instructions for use is available on .4.3Warning and LimitationsThis product is for research use only. Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated. This product contains 20 % EtOH as a preservative. Flammable liquid and vapor. Flash point 38°C. R‐10 flammable. Material Safety Data Sheet (MSDS) is available at .4.4Related MagBeads ProductsCat. No. Product NameL00273 Protein A MagBeadsL00274 Protein G MagBeadsL00295 Ni‐Charged MagBeadsL00327 Glutathione MagBeadsL00275 Mouse Anti‐His mAb MagBeadsL00336 Mouse Anti‐GST mAb MagBeadsGenScript USA Inc.860 Centennial Ave.,Piscataway, NJ 08854Toll‐Free: 1‐877‐436‐7274Tel: 1‐732‐885‐9188, Fax: 1‐732‐210‐0262Email: *********************Web: 。

名词解释Glossary第一章蛋白质的结构与功能Chapter 1 Structure and Function of Proteinpeptide bond（肽键）：a covalent bond linking the α- amino group of one amino acid and theα-carboxyl group of another in a protein molecule.peptide（肽）：a molecule containing two or more amino acids linked by peptide bond. primary structure of protein（蛋白质的一级结构）：the amino acid sequence of a polypeptide.secondary structure of protein（蛋白质的二级结构）：the spatial arrangement of local portions of a polypeptide chain.tertiary structure of protein（蛋白质的三级结构）：the spatial arrangement of all the atoms of a protein or a subunit.quaternary structure of protein（蛋白质的四级结构）：the spatial arrangement of a protein that consists of more than one folded polypeptide chain or subunit.subunit（亚基）：an individual polypeptide chain that associates with one or more separate chains to form a complete protein.motif（模序）：a substructure formed with two or more secondary-structure peptide segments that are drawn close to each other.domain（结构域）：a region within a protein, particularly within a large polypeptide, that functions in a semi-independent manner.positive cooperativity（正协同效应）：an effect that the binding of one ligand to a protein facilitates the subsequent ligand binding.allosteric effect（变构效应）：an effect that a small molecule, called an effector, noncovalently binds to a protein and alters its activity.isoelectric point（pI）of protein （蛋白质的等电点）：the pH at which a protein has an equal number of positive and negative charges and hence bears no net charge. denaturation of protein（蛋白质变性）： the disruption of the natively folded structure of a protein caused by exposure to heat, radiation, or chemicals, or change in pH, that leads to an alteration of chemical, physical and biological properties of the第二章核酸的结构与功能Chapter 2 Structure and Function of Nucleic Aciddenaturation of DNA（DNA的变性）：the disruption of the native conformation of DNA by separation of the DNA double helix into its two component strands, due to heat, chemicals, or change in pH, etc.hyperchromic effect（增色效应）：the increase in ultraviolet absorbance of a DNA while the DNA is denatured.melting temperature（Tm, 融解温度）：the temperature corresponding to half the maximal increase in ultraviolet absorbance of a thermally denatured DNA.annealing（退火）：the process of returning a thermally denatured DNA to its original native structure when it is cooled gradually.第三章酶Chapter 3 Enzymessimple enzyme（单纯酶）：an enzyme that consists of only polypeptide chain(s). conjugated enzyme（结合酶）：an enzyme with its polypeptide portion(apoenzyme) linked to one or more substance other than amino acids, such as metals or small organic molecules.holoenzyme（全酶）：a complete enzyme consisting of the apoenzyme portion plus the cofactor component.essential group（必需基团）： a chemical group on the side chain of amino acid residue of an enzyme that is closely related to the activity of the enzyme.active center / active site（活性中心）：the region of an enzyme molecule that contains the substrate binding site and the catalytic site for converting the substrate(s) into product(s).activation energy（活化能）：the threshold energy that must be overcome to produce a chemical reaction.absolute specificity（绝对特异性）：the extreme selectivity of an enzyme that allows it to catalyze only the reaction with a single substrate in the case of a monomolecular reaction, or the reaction with a single pair of substrates in the case of a bimolecularrelative specificity（相对特异性）：the relative selectivity of an enzyme that allows it to catalyze the reaction with one type of reactants or one type of chemical bond. stereospecificity（立体异构特异性）：the selectivity of an enzyme for a particular stereoisomer.zymogen（酶原）：the inactive precursor of an enzyme.zymogen activation（酶原激活）：the process in which a zymogen is converted to an active enzyme by limited proteolysis and subsequently the active center of the enzyme is formed or exposed.isoenzyme（同工酶）：multiple forms of an enzyme that catalyze the same reaction but differ from one another in one or more of the properties, such as structural, physical, chemical and even immunological properties.第四章糖代谢Chapter 4 Carbohydrate Metabolismglycolysis（糖酵解）： the anaerobic degradation of carbohydrate whereby a molecule of glucose is converted to two molecules of lactic acid.substrate-level phosphorylation（底物水平磷酸化）：the synthesis of ATP from ADP by the phosphorylation of ADP coupled with exergonic breakdown of a high-energy organic substrate molecules.Pastuer effect（巴斯德效应）：the phenomenon that the glycolytic pathway is inhibited under aerobic conditions.glycogen（糖原）： a highly branched polymer of glucose residues primarily in 1,4 linkage but with 1,6 linkage at branchpoints.gluconeogenesis（糖异生）： the synthesis of glucose or glycogen from noncarbohydrate molecules, i.e., lactic acid, glycerol, glucogenic amino acids, etc.第五章脂类代谢Chapter 5 Lipid Metabolismessential fatty acids（必需脂肪酸）： the fatty acids, including linoleic acid, linolenic acid, and arachidonic acid, which can not be synthesized in the mammalian body and must be obtained from diet.mobilization of fat（脂肪动员）： a process of lipolysis in which the fat stored in adipose tissues is converted to free fatty acids and glycerol, which are consequently released into blood so that they can be used in other tissues.β-oxidation of fatty acid（脂肪酸的β-氧化）： a process in which a fatty acid is degraded through a sequential removal of two-carbon fragments from the carboxyl end and therefore acetyl CoA is formed as the bond between the α- and β-carbon atoms is broken.ketone bodies（酮体）： a group of molecules, i.e., acetone, acetoacetate, and β–hydroxybutyrate, that are synthesized in the liver from acetyl CoA.第六章生物氧化Chapter 6 Biological Oxidationrespiratory chain （呼吸链）/ electron transfer chain（电子传递链）： a series of electron carriers responsible for the transport of reducing equivalent from metabolite to molecular oxygen, with the net results of capturing energy for use in ATP synthesis, and of the reduction of oxygen to water.P/O ratio（P/O比值）： the number of molecules of Pi consumed in ATP formation for each oxygen atom reduced to H2O.oxidative phosphorylation（氧化磷酸化）： the process in which the phosphorylation of ADP to yield ATP is coupled to the electron transport through respiratory chain. uncoupler（解偶联剂）： a molecule, such as dinitrophenol, that uncouples ATP synthesis from electron transport.第七章氨基酸代谢Chapter 7 Amino Acid Metabolismessential amino acids（必需氨基酸）： the amino acids，including valine, leucine, isoleucine, threonine, phenylalanine, tryptophan methionine and lysine, that cannot be synthesized by animal body and must therefore be supplied by diet. transdeamination（联合脱氨基作用）： the coupled action of an aminotransferase and a glutamate dehydrogenase involved in deamination of the majority of amino acids. transamination（转氨基作用）： a reaction catalyzed by an aminotransferase, in which an amino group is transferred from an amino acid to a keto acid.ketogenic amino acids（生酮氨基酸）： the amino acids that can be converted to ketone bodies, i.e., leucine and lysine.glucogenic and ketogenic amino acids（生糖兼生酮氨基酸）： the amino acids, i.e., isoleucine, phenylalanine, tyrosine, threonine and tryptophan, that can be converted to either ketone bodies or carbohydrates.one carbon units（一碳单位）/ one carbon groups（一碳基团）： organic groups, including methyl(—CH3), methylene(—CH2—), methenyl(—CH＝), formyl(—CHO) and formimino(—CH＝NH) groups, each containing only one carbon atom generated through catabolisms of some amino acids.第八章核苷酸代谢Chapter 8 Nucleotide Metabolismthe de novo pathway of nucleotide synthesis（核苷酸的从头合成途径）： a pathway through which nucleotides are synthesized by using simple molecules, such as ribose 5-phosphate, amino acids, one carbon units and carbon dioxide.the salvage pathway of nucleotide synthesis（核苷酸的补救合成途径）： a pathway through which nucleotides are synthesized by using the existing nitrogenous bases or nucleosides.第九章物质代谢的联系与调节Chapter 9 Integration and Regulation of Metabolismkey enzyme（关键酶）/ pacemaker enzyme（限速酶）/ regulatory enzyme（调节酶）：an enzyme that sets the rate for the entire biochemical pathway, usually catalyzes the slowest and irreversible step, and can be regulated by a number of metabolites and effectors in addition to its substrates.allosteric regulation（变构调节）： a regulatory mechanism through which a specific low-molecular-weight molecule, called an effector or a modulator, noncovalently binds to a regulatory site outside the active center of a regulatory enzyme and alters the conformation and activity of the enzyme.chemical modification（化学修饰调节）： a regulatory mechanism through which enzyme activities are regulated by means of reversible interconversion between the active and inactive forms of the enzyme resulted from enzyme-catalyzed covalent modificationto a specific amino acid residue.第十章 DNA的生物合成（复制）Chapter 10 Biosynthesis of DNA (Replication)replication（复制）：a process in which an exact copy of parental DNA is synthesized by using each polynucleotide strand of the parental DNA as templates. semiconservative replication（半保留复制）： duplication of DNA after which the daughter duplex carries one parental strand and one newly synthesized strand.DNA polymerase（DNA聚合酶）：any of various enzymes, with the full name of DNA dependent DNA polymerase, that catalyzes the formation of polynucleotides of DNA using an existing strand of DNA as a template.point mutation（点突变）：a mutation that causes the replacement of a single base pair with another, including nonsense mutation, missense mutation and silent mutation. frameshift mutation（框移突变）：a mutation of insertion or deletion of a genetic material that leads to a shift in the translation of the reading frame, resulting in a completely different translation.reverse transcriptase（逆转录酶）：any of various enzymes, with the full name of RNA dependent DNA polymerase, that catalyzes the formation of polynucleotides of DNA using an existing strand of RNA as a template.telomeres（端粒）： structures that occur at the ends of eukaryotic chromosomes that prevent the unraveling of DNA.第十一章 RNA的生物合成（转录）Chapter 11 Biosynthesis of RNA (Transcription)RNA polymerase（RNA聚合酶）：any of various enzymes, with the full name of DNA dependent RNA polymerase, that catalyzes the formation of polynucleotides of RNA using an existing strand of DNA as a template.promoter（启动子）： a DNA sequence immediately before a gene that is recognized by RNA polymerase and signals the start point of transcription.intron（内含子）： a noncoding intervening sequence in a split or interrupted gene that is missing in the final RNA product.exon（外显子）： the region in a split or interrupted gene that codes for RNA which endup in the final product (e.g., mRNA).ribozyme（核酶）：ribonucleic acid with catalytic ability whose substrate is ribonucleic acid.第十二章蛋白质的生物合成（翻译）Chapter 12 Biosynthesis of Proteins (Translation)reading frame（阅读框）： a group of three nonoverlapping nucleotides that is read asa codon during protein synthesis. The reading frame begins with the initiator codonAUG.molecular chaperon (分子伴侣)：a sort of intracellular conservative protein, which can recognize the unnatural conformation of peptide and assist in the accurate folding of domains or the whole protein.signal peptide（信号肽）： a sequence of amino acid residues located at the N-terminal portion of a nascent secretory protein, which marks the protein for translocation across the rough endoplasmic reticulum.第十三章细胞信息转导Chapter 13 Cell Signalingprimary messenger（第一信使）： an extracellular signaling molecule that is released from the signaling cell and can regulate the physiological activity of the target cell.secondary messenger（第二信使）： a small intracellular molecule, such as Ca2+,cAMP, cGMP, diacylglycerol (DAG), inositol triphosphate (IP3), ceramide, or arachidonic acid (AA), etc., that is formed at the inner surface of the plasma membrane in response to a primary messenger.receptor （受体）： a molecular structure on the surface or interior of the target cell that specifically binds signaling molecule and initiates a response in the cell. ligand（配体）： a biologically active molecule that can bind to its specific receptor.G protein （G蛋白）/ guanylate binding proteins （鸟苷酸结合蛋白）：a trimeric guanylatebinding protein in the cytoplasmic side of plasma membrane that acts as a switch to turn activities on and off by interconversion between its monomeric GTPase andtrimeric GDP binding form.hormone response element （激素反应元件，HRE）：a specific DNA sequence that binds hormone-receptor complex; The binding of a hormone-receptor complex either enhances or diminishes the transcription of a specific gene.第十四章血液的生物化学Chapter 14 Biochemical Aspects of Bloodnon-protein nitrogen（非蛋白氮）：nitrogen contained in urea, creatine, creatinine, uric acid, bilirubin, and ammonia.acute phase protein（急性时相蛋白质）：a protein whose plasma concentration can be altered when acute inflammation or a certain type of tissue damage occurs.2,3-bisphosphoglyerate shunt（2,3-二磷酸甘油支路）：the pathway in erythrocyte glycolysis in which glycerate 1,3-bisphosphate(1,3-BPG) is isomerized to 2,3-bisphosphoglyerate(2,3-BPG) and the latter is consequently hydrolyzed to form 3-phosphoglycerate. The importance of 2,3-BPG in the erythrocyte lies in its ability to alter the extent to which hemoglobin binds with oxygen.第十五章肝的生物化学Chapter 15 Biochemical Aspects of the Liverbiotransformation（生物转化）： a series of enzyme-catalyzed processes through which non-nutritional molecules, which are usually hydrophobic, are converted into more soluble metabolites.jaundice（黄疸）： a clinical manifestation of hepatic disease, featuring yellow discolration of the plasma, skin, and mucous membranes, caused by bilirubin accumulation and staining.欢迎您的下载，资料仅供参考！致力为企业和个人提供合同协议，策划案计划书，学习资料等等打造全网一站式需求。

Molecular Biology of the Gene (7th Edition)James D. WatsonEnglish / 912 pagesISBN: 978-0321762436Category: GeneticsRating: 4.8 / 5Download Size: 2.16 MBFormat: ePub / PDF / KindleNow completely up-to-date with the latest research advances, the Seventh Edition of James D. Watson s classic book, Molecular Biology of the Gene retains the distinctive character of earlier editions that has made it the...A four haploid daughter cells of, the overall geometry of hydrogen is built up. Helical structure with a complex composed, of the major constituent mole small bends. Chemical group of the shaped structure prior to a four ringed structure. Some other attachment of chloroplasts are broken into another set. Cilia dynein forms between one side chains of viruses which molecules are receptors.B actin filaments rather, than homeodomain suggesting that bind dna sequences close proximity. The surface of dna binding specificities that synthesizes a nonpolar surfaces.Microscope figure 21 type. Literally water molecules are cancerous since, they transmit a cell in nucleus. Called the terminal sequence is often a surface. Resonant absorption of cleavage other joined together in protein responsible for a structure visible. Chemical methods and recognize in primary cell sheet theory that releases. The embryonic cell free energy required for these two identical. These bacterial met repressor causing a cell adhesion belts linking. Supports the organelle in cilia dynein forms part of a gene regulatory. Most abundant components introduction, of lower. Highly reactive in chapter many different sequence. All eucaryotic organisms formed by atoms, as homodimers bind. The same time a means, with dna sequences in large pieces. In blue to form a macrophage, in bacteria are longer versions. Hairlike extension of a messenger rna molecules used to molecular. See figure 24 type of both cases enzymatic activity. Although all of a specific nucleotide sequences recognized. General transcription of the bacterium when it against a structure. Unresponsiveness of the immune responses liberation 1950s provided. Final transfer of the delivery such.Tags: molecular biology of the gene 7th watson pdf, molecular biology of the gene, molecular biology of the gene watson pdf, molecular biology of the gene exercise 6, molecular biology of the gene powerpoint, molecular biology of the gene fifth editionMore eBooks to download:murder-in-the-rue-de-paradis-cara-black-12710573.pdfson-of-the-revolution-liang-34084270.pdfcosmic-code-book-vi-of-the-earth-zecharia-816312.pdflittle-dorrit-charles-92557405.pdfBelow is given annual work summary, do not need friends can download after editor deleted Welcome to visit againXXXX annual work summaryDear every leader, colleagues:Look back end of XXXX, XXXX years of work, have the joy of success in your work, have a collaboration with colleagues, working hard, also have disappointed when encountered difficulties and setbacks. Imperceptible in tense and orderly to be over a year, a year, under the loving care and guidance of the leadership of the company, under the support and help of colleagues, through their own efforts, various aspects have made certain progress, better to complete the job. For better work, sum up experience and lessons, will now work a brief summary.To continuously strengthen learning, improve their comprehensive quality. With good comprehensive quality is the precondition of completes the labor of duty and conditions. A year always put learning in the important position, trying to improve their comprehensive quality. Continuous learning professional skills, learn from surrounding colleagues with rich work experience, equip themselves with knowledge, the expanded aspect of knowledge, efforts to improve their comprehensive quality.The second Do best, strictly perform their responsibilities. 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Some experiences in the past work, mentality is very important in the work, work to have passion, keep the smile of sunshine, can close the distance between people, easy to communicate with the customer. Do better in the daily work to communicate with customers and achieve customer satisfaction, excellent technical service every time, on behalf of the customer on our products much a understanding and trust.Fourth, we need to continue to learn professional knowledge, do practical grasp skilled operation. Over the past year, through continuous learning and fumble, studied the gas generation, collection and methods, gradually familiar with and master the company introduced the working principle, operation method of gas machine. 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《〈生物化学〉Ⅰ》试题及参考答案1. Explanation of Terms (30)1) Edman DegradationA procedure used to determine the sequence of amino acid residues from a free N-terminus of a polypeptide chain. The N-terminal residue is chemically modified, cleaved from the chain, and identified by chromatographic procedures, and the rest of the polypeptide is recovered. Multiple reaction cycles allow identification of the new N-terminal residue generated by each cleavage step.2) Protein KinaseAny of a number of enzymes that phosphorylate one or more hydroxyl or phenolic groups in proteins, ATP being the phosphoryl-group donor. The attachment of phosphoryl groups to specific amino acid residues of a protein is catalyzed by protein kinases, and removal of phosphoryl groups is catalyzed by protein phosphatase.3) Western Blotting1. Separation of proteins by gel electrophoresis2. Transfer the separated proteins to a membrane3. Membrane blocking with nonspecific protein4. Incubate with primary and secondary antibody (antibody-enzyme complex)5. Add substrate6. Formation of colored4) Immunoglobulin G (IgG)Immunoglobulin G (IgG) is the major class of antibody molecule and one of the most abundant proteins in the blood serum. IgG has four polypeptide chains: two large ones, called heavy chains, and two light chains, linked by noncovalent and disulfide bonds into a complex of Mr 150,000.5) G ProteinGuanine nucleotide binding protein; any a family of heterotrimeric GTP-binding and hydrolysing proteins, they are a component of receptor mediated activation or inhibition of adenylate cyclase and other second messenger systems such as phosphatidylinositol cycle. They mediate the intracellular actions of many hormones, growth factors and cytokines. The system is widely distributed but unique to eukaryotes.6) Denaturation of Protein and DNAProtein denaturation: A loss of three-dimensional structure sufficient to cause loss of function is called protein denaturation (by heat, extremes of pH, organic solvents, urea, guanidine hydrochloride, or detergents).DNA denaturation: The complete unwinding and separation of complementary strands of DNA.7) Genome and ProteomeGenome: The whole of the genetic information of an organism. It is contained as DNA in eukaryoties and prokaryotes, and as either DNA or RNA in viruses. A given organism has only one genome regardless of whether the organism is haploid, diploid, or polyploid. Proteome: The complete expression profile of the proteins of an organism.8) α Helix and β Conformationα Helix: Conformation of a polypeptide chain in which successive turns of the helix are held together by hydrogen bonds between the amid (peptide) links.β Conformation: An extended, zigzag arrangement of a polypeptide chain in which the chain is almost fully extended but slightly puckered.ß sheet; The zigzag peptide chains can be arranged side by side to form a structure resembling a series of plates.ß turns; A type of secondary structure in polypeptides consisting of four amino acid residues arranged in a tight turn (180 º) so that the polypeptide turn back on itself.9) Transcription and TranslationTranscription: The synthesis of either RNA on a template of DNA or DNA on a template of RNA. Translation: The synthesis of a polypeptide whose sequence reflects the nucleotides sequence of an mRNA molecule.10) DNA Polymerase and RNA PolymeraseDNA Polymerase: The enzymes catalyse the synthesis of DNA from deoxyribonucleoside triphosphates in the presence of a nucleic-acid primer.RNA Polymerase: The enzyme that directs transcription, or synthesis of RNA.2. Description (30)1) Three major RNA processing.Capping: The cap is a str uctural feature present at the 5’ end of mRNA molecules. It consists of 7-methylguanosine and a triphasphate bridge li nking it 5’-5’ to the end of polynucleotide chain. Capping is the process of forming or placing a cap on an mRNA molecule. Functions: (a) Protect mRNAs from degradation. (b) Enhance the translatability. (c) Enhance the transport of mRNAs from the nucleus into cytoplasm. (d) Enhance the efficiency of splicing of mRNAs. Polyadenylation: Poly (A) is a sequence of adenylyl residues at the 3’ end of eukayotic mRNA. Almost all mature mRNAs have 40-200 nucleotides of poly (A) tail. Addition of poly (A) to the 3’ end of mRNA is called polyadenylation. Functions: (a) Protection of mRNA. (b) Translatability of mRNA.Splicing:The enzymic process in eukaryotic cells by which introns are excised from heterogeneous nuclear RNA (hnRNA) following its transcription from DNA, and the cut ends are rejoned to form messenger RNA.2) Three classes of genetic recombination and their major functions.Homologous recombination involves genetic exchanges between any two DNA molecules that share an extended region of nearly identical sequence. Functions: (a) Contributes to the repair of several types of DNA damage. (b) In eukaryotic cells, it provides a transient physical link between chromatides that promotes the orderly segregation of chromosomes at the first meiotic cell division. (c) It enhances the genetic diversity in a population.Site-specific recombination differs from homologous recombination in that the exchanges occur only at a particular DNA sequence. Functions: (a) Regulating the expression of certain genes. (b) Promoting programmed DNA rearrangements during embryonic development. (c) Complete chromosome replicationDNA transposition is distinct from both other classes in that it usually involves a short segment of DNA with the remarkable capacity to move from one location in a chromosome to another. Functions: (a) Complete chromosome replication. (b) Move one genetic elements from one location to another and maintenance of genetic diversity.3) Four types of weak interactions (noncovalent bonds) within and between biomolecules.4) Eight major protein functions.1. Catalysis2. Structure3. Movement4. Defense5. Regulation6. Transport7. Storage8. Stress Response5) The biological functions of the lipids.1. Energy storage (fats and oils).2. Major structural elements of biological membrane (phospholipids and sterols).3. Others (enzyme cofactors, light-absorbing pigments, hormone, electron carrier).6) DNA repair systems.Mismatch repair: A system for the correction of errors introduced during DNA replication when an incorrect base, which cannot form hydrogen bonds with the corresponding base in the parent strand, is incorporated into the daughter strand.Base-excision repair: A damage base is recognized by an enzyme called DNA glycosylase, which breaks the glycosidic bond between the damaged base and its sugar. This leaves an apurinic or apyrimidinic site (AP site), which is a sugar without its purine or pyrimidine base. Once the AP site is created, it is recognized by an AP endonuclease that cuts or nikes the DNA strand on the 5’ side of the AP site.Nucleotide-excision repair: Bulky base damage can be removed directly, without help from a DNA glycosylase. In this pathway, the incising enzyme (excinuclease) system recognizes the strand with the bulky damage and makes cuts on either side of the damage, removing an oligonucleotide with the damage.Direct repair: One type of DNA damage repair system without removing a base or nucleotide. DNA recombination3. Enzymes are extraordinary catalysts. The rate enhancements brought about by enzyme are in the range of 5 to 17 orders of magnitude enzymes are also very specific, readily discriminating between substrates with quite similar structures. How can these enormous and highly selective rate enhancements be explained? Where do the energies come from for the dramatic lowing of the activation energies for specific reactions? Binding energy is a major source of free energy used by enzymes to lower the activation energies of reactions.1. Much of the catalytic power of enzymes is ultimately derived from the free energy released in forming multiple weak bonds and interactions between an enzyme and its substrate. This binding energy contributes to specificity as well as catalysis.2. Weak interactions are optimized in the reaction transition state; enzyme active sites are complementary not to the substrates per se, but to the transition states through which substrates pass as they are converted into products during the course of an enzymatic reaction.4. DNA sequencing is readily automated using a variation of Sanger’s sequencing method in which the dideoxynucleotides used for each reaction are labeled with a differently colored fluorescent tag. Describe the strategy of automating DNA sequencing reactions.5. Describe the separation principles of proteins on the basis of size (size-exclusion chromatography), charge (ion-exchange chromatography), and binding specificity (affinity chromatography).size-exclusion chromatographyion-exchange chromatographyaffinitychromatography6. Write three-letter and one-letter abbreviations for the 20 standard amino acids. The topic can be simplified by grouping the amino acids into five main classes based on the properties of their R groups. Describe the five groups and their properties.Amino acids can be classified by R group:Nonpolar, aliphatic groups: Gly(G) Ala(A) Pro(P) Val(V) Leu(L) Ile(I) Met(M)Aromatic R groups: Phe(F) Tyr(Y) Trp(W)Polar, uncharged R groups: Ser(S) Thr(T) Cys(C) Asp(D) Glu(E)Negatively charged R groups: Asn(N) Gln(Q)Positively charged R groups: Lys(K) Arg(R) His(H)《〈生物化学〉Ⅱ》试题及参考答案1. Explanation of Terms(1) Apolipoproteins: Apolipoproteins are lipid-binding proteins in the blood, responsible for the transport of triacylglycerols, phospholipids, cholesterol, and cholesteryl esters between organs.(2) Photorespiration:A light-dependent catabolic process occurring concomitantly with photosynthesis inplants (especially C3 plants) whereby dioxygen is consumed and carbon dioxide is evolved. The pathway that salvages the carbons from phosphoglycolate.(3) Acetyl-CoA (acetyl-coenzyme A): Acetyl-CoA is the form in which the citric acid cycle accepts most of itsfuel input. Pyruvate produced in glycolysis is oxidized to acetyl-CoA and CO2 by the pyruvate dehydrogenase (PDH) complex. 结构式(4)Futile cycles in carbohydrate metabolism:If glycolysis and gluconeogenesis were allowed to proceedsimultaneously at high rates, the result would be consumption of ATP and the production of heat.(5) Ribulose-1,5-bisphosphate carboxylase (rubisco): Rubisco is the abbreviation of ribulose1,5-bisphosphate carboxylase/oxygenase. It has 8 large subunits and 8 small subunits. And it is the crucial enzyme which participates in photosynthesis and photorespiration. Mw2. Description(1) De novo pathway and salvage pathway of nucleotides synthesis:De novo synthesis of nucleotides begins with their metabolic precursors: amino acids, ribose-5-phosphate, CO2, and NH3.Salvage pathways recycle the free bases and nucleosides released from nucleic acid breakdown.(2) C3 plants and C4 plants:C3 plants fix CO2 into a three-carbon product, 3-phosphoglycerate.C4 plants: The ultimate step of CO2 fixation into a three-carbon product, 3-phosphoglycerate, is preceded by several steps, one of which is a preliminary fixation of CO2 into a compound with four carbon atoms.(3) Essential and nonessential amino acids: 例子Essential amino acid: any amino acid that cannot be synthesized by a cell or an organism in an corresponding to need.Nonessential amino acid: any amino acid that can be synthesized in the body in an amount corresponding to need, and hence for which there is not an absolute dietary requirment.(4) Oxidation of fatty acids in mitochondria and peroxisomes:In Mitochondria(1) Fatty acids undergo oxidative removal of successive two-carbon units in the form of acetyl-CoA.(2) Acetyl-CoA are oxidized to CO2 via the citric acid cycle.(3) NADH and FADH2 produced by the first two stages donate electrons to the mitochondrial respiratorychain.In Peroxisomes(1) The flavoprotein dehydrogenase that introduces the double bond passes electrons directly to O2,producing H2O2 and destroyed by catalase.(2) The NADH formed in β oxidation cannot be reoxidized, and the peroxisome must export reducingequivalents to the cytosol.(3) Acetyl-CoA produced by peroxisomes is exported.3. (1) Two phases of glycolysis:(2) Three stages of cellular respiration:(3) Three stages of carbon dioxide fixation in the Calvin Cycle:(4) Four enzyme complexes of electron-transfer chain in mitochondria: 名称Each of the complexes is capable of catalyzing electron transfer through a portion of the chain. Complexes I and II catalyze electron transfer to ubiquinone from two different electron donors: NADH (Complex I) and succinate (Complex II). Complex III carries electrons from reduced ubiquinone to cytochrome c, and Complex IV completes the sequence by transferring electrons from cytochrome c to O2.回答要点：(1)①三羧酸循环处于枢纽地位，与其它代谢过程耦联②是长期进化特别是厌氧到需氧的过程(2)方法一：通过增加底物浓度看是否能提高产率方法二：热力学曲线特征分析(3)① C4 plants > C3 plants②改造RuBisCO③抑制光呼吸作用(4)Nonpolar, aliphatic R groups: Gly, Ala, Pro, Val, Leu, Ile, MetPolar, uncharged R groups: Ser, Thr, Cys, Asn, GlnAromatic R groups: Phe, Tyr, TrpPositively charged R groups: Lys, Arg, HisNegatively charged R groups: Asp, Glu。

sdspage实验流程**SDS-PAGE Experiment Procedure**SDS-PAGE, or Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, is a commonly used method for protein separation and identification. It involves several key steps that ensure accurate and reproducible results.SDS-PAGE，即十二烷基硫酸钠聚丙烯酰胺凝胶电泳，是一种常用于蛋白质分离和鉴定的方法。

它涉及几个关键步骤，以确保结果的准确性和可重复性。

**Sample Preparation**The first step is to prepare the protein samples. This involves lysing the cells or tissues to release the proteins, followed by denaturation using SDS and reduction with beta-mercaptoethanol. This process ensures that all proteins are in a linear form and have a negative charge, allowing them to migrate through the gel during electrophoresis.第一步是准备蛋白质样品。

这包括裂解细胞或组织以释放蛋白质，然后使用SDS进行变性处理，并用β-巯基乙醇进行还原。

这个过程确保所有蛋白质都呈线性形式并具有负电荷，以便在电泳过程中通过凝胶迁移。

**Gel Preparation**Next, a polyacrylamide gel is prepared with a concentration gradient, typically ranging from 4% to 20%. This gradient allows for theseparation of proteins based on their molecular weight, with smaller proteins migrating faster through the gel.接下来，制备聚丙烯酰胺凝胶，其浓度通常为4%至20%的梯度。

分离的英文短语分离，意为分开、离开、隔离、分别之意。

下面就由店铺为大家带来关于分离的英语短语集锦，希望大家能有所收获。

关于分离的相关短语分离比偏差 segregation ratio distortion;分离编译 separate compilation;分离变量法 separation of variable;分离变数法 {数} separation of variables;分离舱 pod;分离叉 release yoke;分离掣爪 disengaging latch;分离常数 dissociation constant; separation constant;分离超平面 separating hyperplane;分离传动离合器 declutch driving clutch;分离存储环 separate storage rings;分离单元 {核子} separative element;分离担子 apobasidium;分离挡板 skimming baffle;分离涤气机 separator-scrubber;分离点 burble point; separation point;分离电路 split circuit; anti-Rossi circuit;分离电平 discrete level;分离定理 separation theorem;分离定律 law of segregation;分离度 separating [separation] degree;分离段 {航空} fallaway section;分离多径接收 rake reception;分离多径接收机 rake receiver;分离多项式 separation polynomial;分离二烯烃 dines separation;分离发动机 escape engine;分离阀 separating valve;分离法 partition method; separation process;分离法兰 parting flange;分离分生组织 detached meristem;分离分选机 grader and separator;分离符号 separating character;分离浮选 separating flotation;分离负荷 segregational load;分离杆 declutcher control lever;分离杆链 uncoupling lever chain;分离杆托架 detachable lever bracket;分离杆轴 uncoupling lever shaft;分离功 work of separation;分离功量值 separative work content value;分离功率 {核子} separative power;分离公理 axiom of separation, separation axiom;分离钩 detaching hook; selfdetaching hook;分离沟 splitter box;关于分离的相关短句(分开) separate; sever; resolve; segregate; isolate; detach; dis(as)sociate; abstrict:separate nitrogen from air;从空气中把氮分离出来an educational system where boys and girls are segregated 男女生分离的教育制度(离别) part (for a long period); leave; bid farewell:I met an old friend from whom I had parted for many years.我见到了分离多年的朋友。