John M. Walker – författare

The last few years have seen the rapid development of new methodology in the field of molecular biology. New techniques have been regularly intro­ duced and the sensitivity of older techniques greatly improved upon. Developments in the field of genetic engineering in particular have con­ tributed a wide range of new techniques. In Volume 1, published in 1983, we introduced the reader to a selection of the more advanced analytical and preparative techniques which we considered to be frequently used by research workers in the field of molecular biology. In choosing techniques for Volume 1 we obviously had to be selective and were unable to cover as broad a spectrum of techniques as we would have liked. However, the pro­ duction of Volume 2 has allowed us to develop the theme initiated in Volume 1 and also expand to include a wider range of subject areas. As with Volume 1, the majority of chapters relate to nucleic acid method­ ology, but we have also covered immunological methodology and protein 1. Obviously, we purification techniques that were not included in Volume see Volume 2 as simply a continuation of Volume 1. As with Volume 1, a knowledge of certain basic biochemical techniques and terminology has been assumed. However, since many areas of molecular biology are developing at a formidable rate and constantly generating new termin­ ology, a glossary of terms has been included.

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.

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.

The last few years have seen the rapid development of new methodology in the field of molecular biology. New techniques have been regularly intro­ duced and the sensitivity of older techniques greatly improved upon. Developments in the field of genetic engineering in particular have con­ tributed a wide range of new techniques. In Volume 1, published in 1983, we introduced the reader to a selection of the more advanced analytical and preparative techniques which we considered to be frequently used by research workers in the field of molecular biology. In choosing techniques for Volume 1 we obviously had to be selective and were unable to cover as broad a spectrum of techniques as we would have liked. However, the pro­ duction of Volume 2 has allowed us to develop the theme initiated in Volume 1 and also expand to include a wider range of subject areas. As with Volume 1, the majority of chapters relate to nucleic acid method­ ology, but we have also covered immunological methodology and protein 1. Obviously, we purification techniques that were not included in Volume see Volume 2 as simply a continuation of Volume 1. As with Volume 1, a knowledge of certain basic biochemical techniques and terminology has been assumed. However, since many areas of molecular biology are developing at a formidable rate and constantly generating new termin­ ology, a glossary of terms has been included.

This book aims to describe the current state of knowledge and possible future developments in a number of major areas of research into the nature, causes and treatment of cancer. The contributing authors have been encouraged to discuss their subjects at the molecular level. It will become apparent to the reader that considerable developments in the understanding of the fundamental nature of cancer, in molecular terms, are constantly being made. This is particularly the case in the area of oncogene research where differences between tumour and normal cells can now be defined in terms of altered expression of DNA sequences. An understanding of the methods available for detecting cancer, of the process of carcinogenesis and of the means available for treating cancer can only be achieved with a precise knowledge of the basic biochemical and molecular processes involved. Since it is all to easy for the research scientist to become totally absorbed within the specialised area of research in which he is involved, the first chapter is an attempt to encourage a broader field of vision by introducing the clinician''s view of the cancer problem, which illustrates the broad spectrum of basic problems that need to be solved by the cancer researcher.

This book aims to describe the current state of knowledge and possible future developments in a number of major areas of research into the nature, causes and treatment of cancer. The contributing authors have been encouraged to discuss their subjects at the molecular level. It will become apparent to the reader that considerable developments in the understanding of the fundamental nature of cancer, in molecular terms, are constantly being made. This is particularly the case in the area of oncogene research where differences between tumour and normal cells can now be defined in terms of altered expression of DNA sequences. An understanding of the methods available for detecting cancer, of the process of carcinogenesis and of the means available for treating cancer can only be achieved with a precise knowledge of the basic biochemical and molecular processes involved. Since it is all to easy for the research scientist to become totally absorbed within the specialised area of research in which he is involved, the first chapter is an attempt to encourage a broader field of vision by introducing the clinician's view of the cancer problem, which illustrates the broad spectrum of basic problems that need to be solved by the cancer researcher.

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.

John Walker and Ralph Rapley have collected a wide-ranging group of molecular and biochemical techniques that are the most frequently used in medical and clinical research, especially diagnostics. The authors-well-established investigators who run their own research programs and use the methods on a regular basis-outline the practical procedures for using them and describe a variety of pertinent applications. Among the technologies presented are southern and western blotting, electrophoresis, PCR, cDNA and protein microarrays, liquid chromatography, in situ hybridization, karyotyping, flow cytometry, bioinformatics, genomics, and ribotyping. The applications include assays for mutation detection, mRNA analysis, chromosome translocations, inborn errors of metabolism, protein therapeutics, and gene therapy.

John Walker and Ralph Rapley have collected a wide-ranging group of molecular and biochemical techniques that are the most frequently used in medical and clinical research, especially diagnostics. The authors-well-established investigators who run their own research programs and use the methods on a regular basis-outline the practical procedures for using them and describe a variety of pertinent applications. Among the technologies presented are southern and western blotting, electrophoresis, PCR, cDNA and protein microarrays, liquid chromatography, in situ hybridization, karyotyping, flow cytometry, bioinformatics, genomics, and ribotyping. The applications include assays for mutation detection, mRNA analysis, chromosome translocations, inborn errors of metabolism, protein therapeutics, and gene therapy.

Hands-on researchers describe in step-by-step detail 73 proven laboratory methods and bioinformatics tools essential for analysis of the proteome. These cutting-edge techniques address such important tasks as sample preparation, 2D-PAGE, gel staining, mass spectrometry, and post-translational modification. There are also readily reproducible methods for protein expression profiling, identifying protein-protein interactions, and protein chip technology, as well as a range of newly developed methodologies for determining the structure and function of a protein. The bioinformatics tools include those for analyzing 2D-GEL patterns, protein modeling, and protein identification. All laboratory-based protocols follow the successful Methods in Molecular Biology™ series format, each offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.

The Protein Protocols Handbook, Second Edition aims to provide a cross-section of analytical techniques commonly used for proteins and peptides, thus providing a benchtop manual and guide for those who are new to the protein chemistry laboratory and for those more established workers who wish to use a technique for the first time. All chapters are written in the same format as that used in the Methods in Molecular Biology™ series. Each chapter opens with a description of the basic theory behind the method being described. The Materials section lists all the chemicals, reagents, buffers, and other materials necessary for carrying out the protocol. Since the principal goal of the book is to provide experimentalists with a full account of the practical steps necessary for carrying out each protocol successfully, the Methods section contains detailed st- by-step descriptions of every protocol that should result in the successful execution of each method. The Notes section complements the Methods material by indicating how best to deal with any problem or difficulty that may arise when using a given technique, and how to go about making the widest variety of modifications or alterations to the protocol. Since the first edition of this book was published in 1996 there have, of course, been significant developments in the field of protein chemistry.

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.

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.