Methods in Molecular Biology – serie

This second eidtion explores new and updated techniques used to understand solid target-specific models in computational toxicology.  Chapters are divided into four sections, detailing molecular descriptors, QSAR and read-across, molecular and data modeling techniques, computational toxicology in drug discovery, molecular fingerprints, AI techniques, and safe drug design. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.Authoritative and cutting-edge, Computational Toxicology: Methods and Protocols, Second Editon aims to ensure successful results in the further study of this vital field.

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.

For many years I performed tissue culture in large scientific insti- tions that had a great deal of infrastructure. When I set up a tissue l- oratory outside such an infrastructure, however, I found there was a shortage of easily accessible information about the basic needs, reagents, and techniques for establishing such a facility. Much had to be done by trial and error or gleaned from originalpapers. Consequently, I felt that a methods book covering a wide variety of techniques from basic culture to the most sophisticated cell analysis would be a very valuable addition to the scientific literature. In the interim, several useful books (listed in Chapter I of this volume) did appear, but none entirely fitted the bill and some are now somewhat dated. Then, in 1984, the first of the Methods in Molecular Biology volumes from Humana Press was published with its step-by-step recipe approach. This format appealed to me, and so I c- tacted John Walker, the series editor, about including cell culture in this series. The result was that we embarked upon a single volume covering both plant and animal cell culture. Such was the richness of the material that this project soon divided itself into separate volumes on animal cell (Volume 5) and plant cell (Volume 6) culture. In this volume (Volume 5), therefore, we have aimed to describe a variety of basic techniques and culture conditions for a range of cell types.

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.

Biology is the study of living things. The classical approach might be described as holistic and descriptive, whereas the modern molecular - proach aims to be investigative, reductionist, and mechanistic . Genes contain all the information for the structure of all living things ; thus, the understanding of how genes are regulated is an important step toward understanding the nature of living things. The study of gene regulation has been made more tractable by the design of simple expe- mental models in which a single gene can be isolated from the milieu of the organism. The new science of molecular biology has introduced techniques that permit the design of such experimental models. In - sence, the genome of the organism is dissected in such a manner that specific genes may now be introduced into an appropriate cell line . Subsequent analysis of the proteins expressed from the genes under study results in the identification of the regulatory DNA sequences .

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.

The three volumes in Methods in Molecular Biology covering Physical Methods of Analysis (vol. 1, Spectroscopic Methods and Analyses: NMR, Mass Spectrometry, and Metalloprotein Techniques; vol. 2, Optical Spectroscopy and Macroscopic Techniques; vol. 3, Cryst- lographic Methods and Techniques) differ from others in this series in several ways. Each volume covers a group of techniques for the char- terization of biological molecules and their interactions that involve the application of modern techniques of physical chemistry. These techniques by and large do not lend themselves to the "hands-on" approach and cannot usually be carried out by the molecular biologist alone, but most often require collaboration with a specialist. The biologist or biochemist contemplating such a collaboration may feel somewhat at a distance from the experimental work and further isolated by the use of the jargons of analytical and physical chemistry. Physical methods have been used in molecular biology from the earliest days, from simple applications of optical spectroscopy to the complexity of X-ray crystallography, and the full range of these me- ods will be covered over the three volumes. The methods dealt with in this first volume have largely developed from beginnings in small molecule chemistry to the point where they play a valuable role in the characterization of biological macromolecules.

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 is the second of three volumes of Methods in Molecular Biology that deal with Physical Methods of Analysis. The first of these, Spectroscopic Methods and Analyses dealt with NMR spec­ troscopy, mass spectrometry, and metalloprotein techniques, and the third will cover X-ray crystallographic methods. As with the first volume. Microscopy, Optical Spectroscopy, and Macroscopic Techniques is intended to provide a basic understand­ ing for the biochemist or biologist who needs to collaborate with spe­ cialists in applying the techniques of modern physical chemistry to biological macromolecules. The methods treated in this book fall into four groups. Part One covers microscopy, which aims to visualize individual molecules or complexes of several molecules. Electron microscopy is the more familiar of these, while scanning tunneling microscopy is a new and rapidly developing tool. Methods for determining the shapes and sizes of molecules in solution are described in Part Two, which includes chapters on X-ray and neutron scattering, light scattering, and ult- centrifugation. Calorimetry, described in Part Three, provides the means to monitor processes involving thermodynamic changes, whether these are intramolecular, such as conformational transition, or the interactions between solutes or between a solute and its sol­ vent. Part Four is concerned with optical and infrared spectroscopy and describes applications ranging from the measurement of protein concentration by UV absorbance to the analysis of secondary struc­ ture using circular dichroism and Fourier-transform infrared spec­ troscopy.

The scientist' s understanding of the cell at the molecular level has advanced rapidly over the last twenty years. This improved understa- ing has led to the development of many new laboratory methods that increasingly allow old problems to be tackled in new ways. Thus the modern scientist cannot specialize in just one field of knowledge, but must be aware of many disciplines. To aid the process of investigation, the Methods Molecular Biology series has brought together many protocols and has highlighted the useful variations and the pitfalls of the different methods. However, protocols frequently cannot be simply taken from the shelf. Thus the starting sample for a chosen protocol may be unavailable in the correct state or form, or the products of the procedure require a different sort of processing. Therefore the scientist needs more detailed information on the nature and requirements of the enzymes being used. This information, though usually available in the literature, is often widely dispersed and frequently occurs in older volumes of journals; not everyone has comprehensive library facilities available. Also many scientists searching out such information are not trained enzymologists and may be unaware of some of the parameters that are important in a specific enzyme reaction.

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.

Protocols in Molecular Parasitology offers a wide range of experi­ mental protocols, each written by experts, to research workers interested in exploring the molecular aspects of parasitology. This is a field that has expanded enormously in scope and potential over the last decade, although it is as yet significantly underrepresented in the literature out­ side of academic journals. The need for manuals such as this one stems in part from the increasing difficulty of reproducing experimental tech­ niques from reports in standard journal papers, where pressures on space usually result in highly abbreviated descriptions. The "Notes" sections of the following chapters are particularly aimed at counteracting this problem. Since the emphasis of the book is on aspects of molecular analysis, it was possible to devote only a limited amount of space to describing protocols for culture and isolation of the primary parasitic material. Therefore, of the numerous parasitic systems that are currently being investigated in laboratories around the world, only techniques for c- turing the "big five" are described here, since they collectively account for the great bulk of research in this field. It is also possible that some minor front-end modifications to certain protocols may be necessary, depending on exactly which parasite is being studied.

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.

DNA sequencing has become increasingly efficient over the years, resulting in an enormous increase in the amount of data gen- ated. In recent years, the focus of sequencing has shifted, from being the endpoint of a project, to being a starting point. This is especially true for such major initiatives as the human genome project, where vast tracts of DNA of unknown function are sequenced. This sheer volume of available data makes advanced computer methods ess- tial to analysis, and a familiarity with computers and sequence ana- sis software a vital requirement for the researcher involved with DNA sequencing. Even for nonsequencers, a familiarity with sequence analysis software can be important. For instance, gene sequences already present in the databases can be extremely useful in the design of cloning and genetic manipulation experiments. This two-part work on Analysis of Data is designed to be a practical aid to the researcher who uses computers for the acquisition, storage, or analysis of nucleic acid (and/or p- tein) sequences. Each chapter is written such that a competent sci- tist with basic computer literacy can carry out the procedure successfully at the first attempt by simply following the detailed pr- tical instructions that have been described by the author. A Notes section, which is included at the end of each chapter, provides advice on overcoming the common problems and pitfalls sometimes enco- tered by users of the sequence analysis software. Software packages for both the mainframe and personal computers are described.

When first conceived, not only was the aim of Protocols for Oligo­ nucleotides and Analogs to provide wide coverage of the ohgonuc- otide chemistry field for readers who are well versed within the field, but also to give investigators just entering into the field a new perspective. The very first book on this topic was edited and published by Michael Gait in 1984, in whose laboratory I encountered the newer aspects of oligonucleotide chemistry. Since then, oligonucleotide research has developed to such an extent that its uses extend far beyond basic studies, and now find wide application throughout clinical science as well. Until recently, the major application of oligonucleotides has been in the area of DNA-based diagnostic and "antisense oligonucleotid- based therapeutic approaches. However, oligonucleotides are now also being used as therapeutic agents and are thus frequently found in clinical trials in humans. Synthesis of unmodified oligonucleotides using automated synthe­ sizers has become a common practice in numerous laboratories. How­ ever, improvements on the existing techniques and the introduction of ever newer methods for oligonucleotide synthesis is constantly driving ahead in the leading research laboratories. And several new oligonucle­ otide analogs have been synthesized and studied for their individual prop­ erties in recent years. The present volume strives to bring the readers the most up-to-date information on the newest aspects of synthesis of oligo­ nucleotides and their analogs. A separate volume covers synthesis of oligonucleotide conjugates, along with most of the analytical techniques presently used for analysis of oligonucleotides.

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.

It is now twenty years since Cohen and Boyer's first steps into DNA cloning. In the time since then, there has been an ever increasing acc- eration in the development and application of the cloning methodology. With the recent development of the polymerase chain reaction, a second generation of the technology has been born, enabling the isolation of DNA (and in particular, genes) with little more information than the p- tial knowledge of the sequence. In fact, DNA sequencing is now so advanced that it can almost be carried out on the industrial scale. As a consequence of these advances, it now appears feasible to sequence whole genomes, including one the size of the human. What are we going to do with this information? The future of basic molecular biology must lie in the ability to analyze DNA (and especially the genes within it) starting at the DNA level. It is for these problems that Protocols for Gene Analysis attempts to offer solutions. So you have a piece of DNA, possibly a gene--what do you do next? The first section of this book contains a number of "basic" te- niques that are required for further manipulation of the DNA. This s- tion is not intended to be a comprehensive collection of methods, but merely to serve as an up-to-date set of techniques. I refer you to other volumes in the Methods Molecular Biology series for further rec- binant DNA techniques.

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.

Peptide synthesis has emerged as one of the most powerful tools in biochemical, pharmacological, immunological, and biophysical la- ratories. Recent improvements include general solid-phase method- ogy, new protecting groups, and automated equipment. These advances have allowed the facile synthesis of increasingly more complex p- tides. Many of these new and improved methods for the synthesis of peptides and peptide-related substances have been reported in various publications, but never compiled in a convenient handbook. Like other volumes in this series, Peptide Synthesis Protocols concentrates on the practical aspects of these procedures, providing the researcher with detailed descriptions and helpful tips about potential problems. This volume is not intended to serve as a basic guide to standard Merrifie- type solid-phase strategy, but rather to provide the researcher with some of the most recent applications in the field of peptide science. A c- panion volume, Peptide Analysis Protocols, will detail methodology for the charaterization of new synthetic peptides. Development of new methods and applications has continued actively even as this volume was in preparation. Owing to the number of contributors to this volume, it was necessary to establish a cutoff for publication purposes. We feel that all of the protocols presented are timely and up-to-date. Several promising new strategies, such as allyloxycarbonyl-based syntheses, were being developed at the time this volume was in the editing stages and will be included in future editions.

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.

DNA sequencing has become increasingly efficient over the years, resulting in an enormous increase in the amount of data gener­ ated. In recent years, the focus of sequencing has shifted, from being the endpoint of a project, to being a starting point. This is especially true for such major initiatives as the human genome project, where vast tracts of DNA of unknown function are sequenced. This sheer volume of available data makes advanced computer methods essen­ tial to analysis, and a familiarity with computers and sequence analy­ sis software a vital requirement for the researcher involved with DNA sequencing. Even for nonsequencers, a familiarity with sequence analysis software can be important. For instance, gene sequences already present in the databases can be extremely useful in the design of cloning and genetic manipulation experiments. This two-part work on Computer Analysis of Sequence Data is designed to be a practical aid to the researcher who uses computers for the acquisition, storage, or analysis of nucleic acid (and/or pro­ tein) sequences. Each chapter is written such that a competent scien­ tist with basic computer literacy can carry out the procedure successfully at the first attempt by simply following the detailed prac­ tical instructions that have been described by the author. A Notes section, which is included at the end of each chapter, provides advice on overcoming the common problems and pitfalls sometimes encoun­ tered by users of the sequence analysis software.

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