Methods in Molecular Biology – serie

In recent years there has been a tremendous increase in our understanding of the functioning of the cell at the molecular level. This has been achieved in the main by the invention and development of new methodology, parti- larly in that area generally referred to as "'genetic en- neering". While this revolution has been taking place in the field of nucleic acids research, the protein chemist has at the same time developed fresh methodology to keep pace with the requirements of present day molecular bi- ogy. Today's molecular biologist can no longer be content with being an expert in one particular area alone. He/she needs to be equally competent in the laboratory at h- dling DNA, RNA, and proteins, moving from one area to another as required by the problem he/she is trying to solve. Although many of the new techniques in molecular biology are relatively easy to master, it is often difficult for a researcher to obtain all the relevant information nec- sary for setting up and successfully applying a new te- nique. Information is of course available in the research l- erature, but this often lacks the depth of description that the new user requires. This requirement for in-depth pr- tical details has become apparent by the considerable - mand for places on our Molecular Biology Workshops held at Hatfield each summer.

In recent years there has been a tremendous increase in our understanding of the functioning of the cell at the molecular level. This has been achieved in the main by the invention and development of new methodology, parti- larly in that area generally referred to as "genetic en- neering". While this revolution has been taking place in the field of nucleic acids research, the protein chemist has at the same time developed fresh methodology to keep pace with the requirements of present day molecular bi- ogy. Today's molecular biologist can no longer be content with being an expert in one particular area alone. He/she needs to be equally competent in the laboratory at h- dling DNA, RNA, and proteins, moving from one area to another as required by the problem he/she is trying to solve. Although many of the new techniques in molecular biology are relatively easy to master, it is often difficult for a researcher to obtain all the relevant information nec- sary for setting up and successfully applying a new te- nique. Information is of course available in the research l- erature, but this often lacks the depth of description that the new user requires. This requirement for in-depth pr- tical details has become apparent by the considerable - mand for places on our Molecular Biology Workshops held at Hatfield each summer.

In recent years there has been a tremendous increase in our understanding of the functioning of the cell at the molecular level. This has been achieved in the main by the invention and development of new methodology, parti- larly in that area generally referred to as "'genetic en- neering". While this revolution has been taking place in the field of nucleic acids research, the protein chemist has at the same time developed fresh methodology to keep pace with the requirements of present day molecular bi- ogy. Today's molecular biologist can no longer be content with being an expert in one particular area alone. He/she needs to be equally competent in the laboratory at h- dling DNA, RNA, and proteins, moving from one area to another as required by the problem he/she is trying to solve. Although many of the new techniques in molecular biology are relatively easy to master, it is often difficult for a researcher to obtain all the relevant information nec- sary for setting up and successfully applying a new te- nique. Information is of course available in the research l- erature, but this often lacks the depth of description that the new user requires. This requirement for in-depth pr- tical details has become apparent by the considerable - mand for places on our Molecular Biology Workshops held at Hatfield each summer.

In recent years there has been a tremendous increase in our understanding of the functioning of the cell at the molecular level. This has been achieved in the main by the invention and development of new methodology, parti- larly in that area generally referred to as "genetic en- neering". While this revolution has been taking place in the field of nucleic acids research, the protein chemist has at the same time developed fresh methodology to keep pace with the requirements of present day molecular bi- ogy. Today's molecular biologist can no longer be content with being an expert in one particular area alone. He/she needs to be equally competent in the laboratory at h- dling DNA, RNA, and proteins, moving from one area to another as required by the problem he/she is trying to solve. Although many of the new techniques in molecular biology are relatively easy to master, it is often difficult for a researcher to obtain all the relevant information nec- sary for setting up and successfully applying a new te- nique. Information is of course available in the research l- erature, but this often lacks the depth of description that the new user requires. This requirement for in-depth pr- tical details has become apparent by the considerable - mand for places on our Molecular Biology Workshops held at Hatfield each summer.

Inrecent years therehasbeen atremendousincreaseinour understandingofthe functioningofthe cellat the molecular leveL This has been achieved in the main by the invention and developmentof new methodology, particularlyin that area generally referred to as "genetic engineering. " Al- though this revolution has been taking place in the field of nucleic acids research, the protein chemist has at the same timedevelopedfresh methodologytokeeppace with the re- quirements ofpresent-daymolecularbiology. Today's mo- lecularbiologistscannolongerbecontentwithbeingexperts inoneparticulararea alone. Theyneedtobeequallycompe- tentin the laboratory at handling DNA, RNA,and proteins movingfrom one area toanotherasrequiredby theproblem thatisbeing solved. Althoughmanyof thenewtechniques in molecularbiologyare relativelyeasy tomaster, itisoften difficult for a researcher to obtain all the relevant informa- tionnecessaryforsettingupandsuccessfullyapplyinganew technique. Informationisofcourse availablein the research literature, but this often lacks the depth of description that the new user requires.This requirement for in-depth prac- tical details has become apparent by the considerable de- mand for places on our Molecular BiologyWorkshops held at Hatfield each summer. Volume 1of this series describedpracticalprocedures for a range of protein techniques frequently used by research workers in the field of molecular biology. Because of the limitations on length necessarily inherent in producing any v vi Preface book, one obviouslyhad to be selective in the choiceof titles forVolume1. TheproductionofVolume 3,therefore,allows the development of the theme initiated in Volume 1. This volumecontains afurther selection ofdetailed protocols for arangeofanalyticalandpreparativeproteintechniques,and should be seen as a continuation of Volume 1. Companion Volumes2and4provideprotocols fornucleic acid method- ology. Each methodisdescribedby an authorwhohas regularly used the technique in his or her ownlaboratory.

Animal Cell Culture, the latest volume in Humana's highly successful Methods in Molecular Biology series, provides detailed practical techniques for the culture of a broad spectrum of basic cell cell types. Chapters offer hands-on methods for creating mammalian fibroblastic cell cultures and maintaining culture conditions for epithelial, neuronal, and hematopoietic cells among others. Attention is given to the diversity of culture media and extracellular matrices needed to maintain the differentiated functions of the cultured cells. The book's special strength lies in its descriptions of culture techniques for both living and fixed cells. Chapters cover techniques such as: cinematographic analysis in situ mRNA hybridization immunofluorescence immunoelectron microscopy somatic cell hybridization DNA transformation insect cell culture creation of hybridoma cell lines monoclonal antibody techniques new, specialized methodologies. A useful appendix lists the most commonly used culture media. Comprehensive in scope and coverage, and thoroughly up-to-date, Pollard and Walker's unique handbook on ANIMAL CELL CULTURE is an indispensable resource for both the novice and the seasoned expert.

Plant Cell and Tissue Culture continues the high standards of Humana's Methods in Molecular Biology series. Its step-by-step approach (a hallmark of the series) is applied to a wide range of basic laboratory techniques and culture conditions appropriate to plant cells. Because of the diversity of cell types species and culture methods much of this volume is devoted to the culture of particular cell types and to the regeneration of these cells into whole plants. Special attention is also given to the genetic modification of plants as well as to the economic significance of plant products. Chapters cover a wide range of topics and techniques including: tissue culture media and selection cryopreservation callus culture techniques organ culture embryogenesis batch culture large-scale culture hormonal control fertilization techniques gene transfer cell immobilization production systems cell product purification DNA expression electrofusion of plant cells mutant selection mutagenesis techniques automation transfer of nuclei protoplast culture media analysis micropropagation. A detailed appendix lists the formulas for the most commonly employed plant cell media.Comprehensive easy to follow and a pleasure to use Pollard and Walker's Plant Cell and Tissue Culture is an essential tool for everyone--at all levels of proficiency and experience--involved in plant culture.

Murray's new handbook on Gene Transfer and Expression Protocols sets forth both current and new methodologies in a clear concise easy-to-follow manner following the successful formula of the classic volumes in Humana's Methods in Molecular Biology series. Each chapter is devoted to a thorough exposition of a single technique. An Introduction explains the significance of the protocol and provides background information. A Materials section lists all the requirements for the technique discussed. A Methods section details the procedure in a step-by-step protocol. A Notes section alerts the reader to pitfalls that may be encountered as well as alternatives that may be used for successful completion of the experiment. Each technique is designed to guarantee optimum results. This volume is an outstanding new benchtop manual that provides protocols for introducing an isolated gene into a cell line using various transfection techniques.Topics and techniques include: viral vectors reporter genes analysis of steady-state level of transcription assay for newly initiated transcriptional complexes immunocytological techniques assay of structure and replication state of transfected genes overall strategy for defining regulatory sequences Providing the latest techniques in a fast-paced area Gene Transfer and Expression Protocols is an absolutely essential handbook for everyone involved in cytogenetics cell genetics molecular biology and related fields.

Recombinant viruses provide an efficient mechanism for the transfer and expression of DNA in eukaryotic cells. First, the transfer of DNA by viral infection-utilizing specific cell surface receptors and cellular intern- ization mechanisms-occurs much more readily than DNA transfer via uptake induced by such physical methods as calcium phosphate coprecipitation or electroporation. Second, the novel strategies employed by the virus to express its own genes can then be "hijacked" in the recombinant virus to express the researcher's gene of interest The purpose of Practical Molecular Virology isthus to compile a coll- tion of readily repeatable gene transfer and expression methods from wo- ers expert in the use of a variety of recombinant viral vectors . These include those designed for the production of recombinant antigens, such as pol- virus and yeast Ty-VLPs; those giving very high levels of recombinant protein expression, for example, baculovirus, vaccinia virus, and SV40; and finally viral vectors used for efficient, stable gene transfer to eu otic cells, such as retroviruses and herpesviruses .The first chapter describes the viral life cycle for each virus, and explains how this can be adapted to allow construction of recombinant vectors. Subsequent chapters deal with methods for producing and char- terizing recombinant viruses . I make no apology for the hyperproliferation of chapters dealing with recombinant retroviral methods and applications, since I believe this is clearly proportional to the recent expansion of interest in these techniques.

0ne 0fthe many 1mpact5 0frec0m61nant DNA techn0109y 0ver the 1a5t 15 year5 ha5 6een a 5tr0n91y refre5hed 1ntere5t 1n meth0d5 f0r the 5eparat10n and pur1f1cat10n 0f pr0te1n5.7h15 1ntere5t ha5 enc- pa55ed n0t 0n1y ana1yt1ca1 5eparat10n5, 6ut a150 5ma11- and 1ar9e-5ca1e preparat1ve meth0d5 d1rected t0 60th pure and app11ed re5earch thr0u9h0ut 610109y and 610med1c1ne. Many 0f the new 0r 5u65tant1a11y m0d1f1ed techn14ue5 deve1- 0ped have 6een rep0rted 1n the 11terature, 6ut a 5uff1c1ency 0f deta11ed pract1ca1 he1p 1n e5ta6115h1n9 the5e meth0d5 f0r the f1r5t t1me 1n a new 1a60rat0ry ha5 0ften 6een d1ff1cu1t t0 f1nd. W1th the5e pr061em5 1n m1nd, we expect that Pract1ca1Pr0te1n Chr0mat09raphy, de519ned a5 a key v01ume 1n the Meth0d5 1n M01ecu1ar 810109y 5er1e5, w111 pr0v1de c0nc15e pract1ca1 he1p t0 th05e carry1n9 0ut new techn14ue5 f0r the f1r5t t1me. Each chapter ha5 6een wr1tten 6y expert auth0r5 kn0wn t0 have d1rect and re9u1ar pract1ca1 exper1ence w1th the1r ch05en techn14ue5. 7he 5tructure 0feach chapter 15 de519ned t0 make 1t ea5y f0r a w0rker new t0 the meth0d t0 f0110w 1t t0 an effect1ve c0nc1u510n. An 1nt- duct10n treat5 the the0ry 6eh1nd the meth0d 6e1n9 de5cr16ed.7he Mater1a15 and Meth0d5 5ect10n5 a110w the reader t0 prepare f0r, and then perf0rm techn14ue5 1n a rat10na1 5tepw15e manner. 7he N0te5 5ect10n5 pr0v1de the 50rt 0f6ack9r0und h1nt5 and tr1ck5 that are 50 0ften e55ent1a1 f0r 5ucce55, 6ut are rare1y rep0rted 1n the 11terature.

Although spectroscopic methods are of great use to biological scientists, many researchers lack a specialist's knowledge of these techniques. Spectroscopic Methods and Analyses provides hands-on information on these valuable research tools, emphasizing practical aspects, such as amounts and purity of required samples, limitations and advantages of the technique, and interpretation of the results, thus enabling every molecular biologist - either independently or with the help of a specialist - to evaluate the usefulness of a technique, to plan an original program, and to gain a realistic idea of the resources needed.An indispensable guide for all biochemists, cell and molecular biologists, pharmacologists, immunologists, pathologists, and neuroscientists.

As the editorof Glycoprotein Analysis in BiornedidneI have tried to bring together a comprehensive range ofdetailed protocols that will rendercarbohydratebiochemistryaccessible to all, andnotjust to the dedicated few. I wish to thank the authors, all ofwhom have striven significantly to realize this aim. There is now a growing interest in glycoprotein analysis from researchers in many disciplineswho realize thatprotein glycosylation can no longer be ignored. Oligosaccharide chains of glycoproteins form a diverse arrayofmolecules with an inherentlylarge contentof chemical information that is functional in biological recognition. In addition to theirspecificinteractionswithantibodiesandcarbohydrate binding proteins, the carbohydrate moieties of glycoproteins have profoundly important effects on the conformations, stability, antige- nicity, and function ofthe protein components. Some specialized techniques, Le., NMRand MS, are introduced atthebeginningofthebooksince thesearecapableofdefiningexactly the diversityofoligosaccharide sequences,and are used to detect the growing number ofnew structures as well as to indentiry, in new sce- narios, those previously documented.Together with the chromato- graphic methods of purification and analysis discussed in Chapters 1-12, there now exists a powerful armamentarium for glycoprotein characterization. The more chemical techniques are documented in detail in the earliest chapters, whereas the enzymatic methods of releasing oligosaccharidesfrom protein areintroducedlater. Analysis ofglycoproteinsfrom mammalian, parasitic,bacterial,andviralorigins are each discussed.

This superb survey of the possible applications of physiochemical techniques to the analysis of biological macromolecules is designed for the practicing biochemist or biologist who wants to use the technique but lacks the experience. The contributors emphasize practical aspects, such as constraints on sample quantity, purity, and presentation; the problems of time and expense involved; the problems a technique is best suited to solve; and how the results may be interpreted. Among the methods examined are optical and infrared spectroscopy, microscopy, ultracentrifugation, calorimetry, X-ray and neutron scattering, and light scattering. This book will enable the reader to confidently collaborate with specialists in applying these techniques.

Provides key information on a wide range of enzymes commonly used as tools in molecular biology, helping to minimize the time a scientist spends researching the literature to get reactions to work efficiently and allowing the nonenzymologist to design an experiment. Each chapter gives background information on the enzyme selected and those parameters important in its use, describes both the source and application of the enzyme, and provides details on the size and structure of the protein. Specific parameters essential for achieving an optimized reaction are discussed, along with exemplary practical procedures an protocols.

Expert practitioners combine the traditional basic techniques of membrane isolation and analysis with modern advances in molecular biology, immunology, and clinical sciences to devise analytical techniques that are successful for expert and novice alike. The techniques are used in the isolation and compositional analysis of membranes and address animal, plant, and microbial cells. The topics covered in this first volume include the isolation of subcellular fractions, centrifugation, the extraction and quantitation of lipids, the separation and analysis of proteins, and the isolation and characterization of glycosylated molecules, glycoproteins, proteoglycans, and glycolipids.

Experts present methods and protocols essential for understanding parasites at the molecular level. The protocols cover culturing techniques for the major experimental organisms, methods for isolating and processing nucleic acids and proteins, PCR-based protocols for parasite identification, gene isolation and mutation, antibody-based procedures, chromosome and epitope mapping, flow cytometry, RNA sequencing, and parasite transformation.

Clear, step-by-step instructions provide you with all the techniques used in both classical and molecular cytogenetics. The emphasis is on the use of human material, but the techniques are designed to be readily applicable to other animal chromosomes. Chapters feature methods used in the preparation of chromosomes, in analyzing chromosomes directly at the light-microscope level, and in molecular genetics techniques.

These comprehensive, up-to-date handbooks are designed for those scientists engaged in the computer analysis of sequence data who want hands-on help in using the most important commercial software available, but simply do not have the time to become computer experts. The expert authors guide you through the programs with easy-to-follow, step-by-step instructions. The topics covered include translations of sequences, sequence alignment, phylogenetic trees, analysis of RNA secondary structure, database searching, submission of data to EMBL/GenBank/DDBJ/etc., maintaining sequence projects, and using patterns to analyze protein sequences. Many chapters have been written by world-class authorities in the field, among them R. Staden, M. Gribskov, D. Higgins, W. Pearson, M. Zuker, and G. Barton. Each volume shares five essential chapters concerning the analysis of sequence data, the FASTA program, converting between sequence formats, obtaining software via INTERNET, and the submission of nucleotide sequence data. Part I covers GCG, MicroGenie, PC/GENE, and FASTA programs. Part II covers Staden and Staden Plus, DNA Strider, FASTA, and MacVector programs.

Leading researchers summarize and describe their experience and techniques for synthesizing oligonucleotides and their analogs. The protocols presented cover all DNA analogs that have been studied to date and constitute the single most extensive source of knowledge about DNA chemistry available.

D ring the past ten ears or so, there has been an increasing n mber of p blications describing ario s aspects of oligon cleotide chemistr and the application of oligon cleotides in basic, as ell as applied science. More recentl , ario s oligon cleotide conj gates nthesi ed that are c rrentl being st died for their ha e been s potential diagnostic and therape tic applications. With the introd- tion of ne oligon cleotide conj gates, anal tical methods for the anal sis of these oligon cleotides are also being de eloped and op- mi ed. The present ol me, Protocols for Oligon cleotide Con- gates: S nthesis and Anal tical Techniq es, is designed to bring readers p-to-date on the protocols for the s nthesis of ario s oli- gates and on all the important techniq es for the n cleotide conj nthetic oligon cleotides and their analogs. The s- anal sis of s thetic chemistrof man of these oligon cleotides and their ario s analogs has been co ered in a companion ol me entitledProtocols for Oligon cleotides and Analogs: S nthesis and Properties. The first chapter ofProtocols for Oligon cleotide Conj gates- scribes the protecting gro ps in oligon cleotide s nthesis.

This volume provides a set of protocols for the analysis of cloned genes. A set of techniques is described for the basic manipulation and mutagenesis of the cloned sequences. The rest of the volume is organized into a collection of techniques appropriate to the analytical route to be investigated; these include genome structure, sequence variation, gene expression, and protein function. Finally, a number of methods are described to take the step from the first gene to those with which it interacts.

The first volume in this Methods Molecular Biology series, Proteins (1984), concentrated on basic techniques for the analysis and purification of peptides and proteins. As the series developed, more specialized volumes on proteins were introduced, such as those on Immunochemical Protocols (vol. 10), Practical Protein Chro- tography (vol. 11), Analysis Glycoprotein Biomedicine (vol. 14), Protein-DNA Interactions (vol. 30), Biomembrane Protocols (vols. 19 and 27), Analyses and Methods (vol. 17), and Optical Spectroscopy, Microscopy, and Macroscopic Techniques (vol. 22). Further specialist volumes on peptides, monoclonal antibodies, immunoassays, ELISA, protein engineering, protein stability, mass spectrometry of proteins, automated sequence analysis, and protein NMR are currently in preparation. Since it is now a decade since the initial volume was published, it seems an especially appropriate moment to extensively reorganize, update, and revise the earlier volume.In an attempt to be more c- prehensive in our coverage, this current volume, Basic Protein and Peptide Protocols, is totally committed to basic analytical methods; a planned companion volume will later concentrate on preparative techniques. Those analytical techniques requiring expensive speci- ized instrumentation, such as NMR, mass spectrometry, X-ray cr- tallography, spectroscopy, and automated sequence analysis, are not described here, but in the appropriate specialized volumes listed above.

The first volume in this Methods Molecular Biology series, Proteins (1984), concentrated on basic techniques for the analysis and purification of peptides and proteins. As the series developed, more specialized volumes on proteins were introduced, such as those on Immunochemical Protocols (vol. 10), Practical Protein Chro- tography (vol. 11), Analysis Glycoprotein Biomedicine (vol. 14), Protein-DNA Interactions (vol. 30), Biomembrane Protocols (vols. 19 and 27), Analyses and Methods (vol. 17), and Optical Spectroscopy, Microscopy, and Macroscopic Techniques (vol. 22). Further specialist volumes on peptides, monoclonal antibodies, immunoassays, ELISA, protein engineering, protein stability, mass spectrometry of proteins, automated sequence analysis, and protein NMR are currently in preparation. Since it is now a decade since the initial volume was published, it seems an especially appropriate moment to extensively reorganize, update, and revise the earlier volume.In an attempt to be more c- prehensive in our coverage, this current volume, Basic Protein and Peptide Protocols, is totally committed to basic analytical methods; a planned companion volume will later concentrate on preparative techniques. Those analytical techniques requiring expensive speci- ized instrumentation, such as NMR, mass spectrometry, X-ray cr- tallography, spectroscopy, and automated sequence analysis, are not described here, but in the appropriate specialized volumes listed above.

In Peptide Synthesis Protocols leading authorities assemble in one volume a broad range of state-of-the-art methods for the preparation, purification, and synthesis of peptides. These powerful and useful protocols contain many innovations not previously described, including a compilation of chemical modification procedures and peptide synthesis improvements. The authors describe each procedure in a practical, step-by-step fashion suitable for both the skilled and novice researcher. They also provide troubleshooting tips, alternative ways of doing things, and informative explanations about why certain steps are necessary-aids not usually found in standard journal recipes-all designed to guarantee a significant difference in the outcome of your experiments. Pennington and Dunn's Peptide Synthesis Protocols offers a comprehensive collection of standardized, readily reproducible methods for many critical procedures that will enable scientists in every area of biochemical and biological research to prepare, purify, characterize, and use peptides effectively.

Antibodies are the instraments of immune defense and attack. They can bind small atomic arrays as well as large epitopes with high affinity. Antibody Engineering Protocols presents advanced protocols in the field of antibody engineering, reviews of basic principles and methodology, and a historical perspective on the development of cur- rently held beliefs about antibody structure-function relationships. The topics cover analysis of antibody sequences, three-dimensional structure, delineation of antibody characteristics in polyclonal mixtures, phage display of natural and synthetic antibodies, and antibody catalysis. Ligand recognition by antibodies occurs primarily at a subset of amino acid residues located in the complementarity determining regions (CDRs) found in the light (L) and heavy (H) chain subunits. Specific antibodies are developed by immunization with molecules identical to or related in structure to the target ligand.The immune repertoire from nonimmunized individuals also contains pre-existing specificities that can be selected by screening libraries composed of hybridoma cells or phage particles displaying F domains or indi- vidual variable domains of the light (VJ and heavy (V^^) chains. Ran- dom or site-directed mutagenesis in vitro can be used to refine the pre-existing specificities or produce new specificities de novo. Another level at which new specificities may be generated in vitro is V^ and V^ domain-swapping and CDR-swapping. The former procedure embodies a variation of natural mechanisms of generating antibody diversity. The latter procedure produces new intramolecular CDR combinations not found in nature.

These comprehensive, up-to-date handbooks are designed for those scientists engaged in the computer analysis of sequence data who want hands-on help in using the most important commercial software available, but simply do not have the time to become computer experts. The expert authors guide you through the programs with easy-to-follow, step-by-step instructions. The topics covered include translations of sequences, sequence alignment, phylogenetic trees, analysis of RNA secondary structure, database searching, submission of data to EMBL/GenBank/DDBJ/etc., maintaining sequence projects, and using patterns to analyze protein sequences. Many chapters have been written by world-class authorities in the field, among them R. Staden, M. Gribskov, D. Higgins, W. Pearson, M. Zuker, and G. Barton. Each volume shares five essential chapters concerning the analysis of sequence data, the FASTA program, converting between sequence formats, obtaining software via INTERNET, and the submission of nucleotide sequence data. Part I covers GCG, MicroGenie, PC/GENE, and FASTA programs. Part II covers Staden and Staden Plus, DNA Strider, FASTA, and MacVector programs.

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Leading researchers summarize and describe their experience and techniques for synthesizing oligonucleotides and their analogs. The protocols presented cover all DNA analogs that have been studied to date and constitute the single most extensive source of knowledge about DNA chemistry available.