Year: 2005
Type: article
Abstract: Proteins are like people: they are highly diverse, with very few traits shared by the entire population. Even close relatives often have very different structural (physical) characteristics and behave quite differently. This heterogeneity presents challenges for scientists interested in studying protein structure and function. The need for a readily accessible, updatable reference for protein methods has grown in tandem with the ongoing protein science renaissance driven by advances in molecular biology, genetics, structural biology, and related disciplines. Using recombinant methods, it is now practical to overexpress both normal and reengineered forms of a protein and to create chimeric proteins with unique new properties. The ability to express proteins usually found in low abundance at high levels and to manipulate their amino acid sequences provides unprecedented opportunities to study a vast array of proteins and their associated biological processes (both in vitro and in vivo). The combination of biochemical, biophysical, and high-resolution structural analyses of proteins with manipulation of the associated genes (in cultured cells or an entire mammalian organism) has already led to spectacular advances in many areas of biotechnology and molecular medicine. Because proteins are so diverse, a wide range of techniques must often be considered and tested empirically for a particular protein, and each researcher's arsenal must include both recent innovations and techniques that have been used for many years. Moreover, methods appropriate for purifying and initially characterizing the protein(s) associated with a biological activity of interest are often not suitable for analyzing the corresponding overexpressed recombinant protein. This manual includes both specific detailed protocols as well as strategies for adapting methods to particular projects at hand. It is designed to meet the needs of scientists with little prior experience of protein isolation and characterization, including graduate students and scientists trained in other biological disciplines. At the same time, the broad range of techniques presented here should ensure that even seasoned experts will find new and useful approaches and will benefit from the manual's convenient compilation of standard information and methods that typically must be culled from many sources. The quarterly updating service will allow ongoing expansion of the core volume to include chapters covering critical topics that were deferred due to initial space limitations and to keep pace with the “cutting edge” of developments in this fast-moving field. This publication is available in looseleaf, CD-ROM and online formats. For looseleaf subscribers, binders are provided to accommodate the growth of the manual via the quarterly updates. The looseleaf binder format allows easy insertion of new pages, units, and chapters that are added. The index and table of contents are updated with each supplement. CD-ROM subscribers receive a completely new disc every quarter and should dispose of their outdated discs. The online version is available through institutional license and offers many useful features such as downloadable PDF files of units, enhanced text searching for contents from all Current Protocols titles, real-time updating, and superior color graphics. The material covered in all three versions is identical. Subjects in this manual are organized by chapters and sections, and protocols are contained in units. Units generally describe a method and include one or more protocols with listings of materials, steps and annotations, recipes for unique reagents and solutions, and commentaries on the “hows” and “whys” of the method; there are also “overview” units containing theoretical discussions that lay the foundation for subsequent protocols. Page numbering in the looseleaf version reflects the modular arrangement by unit; for example, page 2.3.5 refers to Chapter 2 (Computational Analysis), UNIT Unavailable (Protein Secondary Structure Prediction), page 5 of that particular unit. Many reagents and procedures are employed repeatedly throughout the manual. Instead of duplicating this information, cross-references among units are used extensively. Cross-referencing helps to ensure that lengthy and complex protocols are not overburdened with steps describing auxiliary procedures needed to prepare raw materials and analyze results. Certain units that describe commonly used techniques and recipes (e.g., gel electrophoresis, ion-exchange chromatography) are cross-referenced in other units that describe their application. Thus, whenever it is necessary to isolate or identify a protein band in a protocol, the appropriate unit in Chapter 10—describing various procedures for gel electrophoresis—is cross-referenced (i.e., UNIT Unavailable). For some widely used techniques (such as dialysis), readers are cross-referenced to APPENDIX 3. Because this publication is first and foremost a compilation of laboratory techniques in protein science, we have not provided extensive instructive material. We have, however, included explanatory information where required to help readers gain an intuitive grasp of the procedures. Chapter 1 provides an overview of strategies for protein purification and characterization, to help the reader plan for all aspects of an experimental study. Some subsequent chapters begin with special overview units that describe the state of the art of the topic matter and provide a context for the procedures that follow. Chapter and unit introductions describe how the protocols that follow connect to one another, and annotations to the actual protocol steps describe what is happening as a procedure is carried out. Finally, the Commentary that closes each protocol unit describes background information regarding the historical and theoretical development of the method, as well as alternative approaches, critical parameters, troubleshooting guidelines, anticipated results, and time considerations. All units contain cited references and many indicate key references to inform users of particularly useful background reading, original descriptions, or applications of a technique. Many units in the manual contain groups of protocols, each presented with a series of steps. The basic protocol is presented first in each unit and is generally the recommended or most universally applicable approach. Alternate protocols are provided where different equipment or reagents can be employed to achieve similar ends, where the starting material requires a variation in approach, or where requirements for the end product differ from those in the basic protocol. Support protocols describe additional steps that are required to perform the basic or alternate protocols; these steps are separated from the core protocol because they might be applicable to other uses in the manual, or because they are performed in a time frame separate from the basic protocol steps. Reagents required for a protocol are itemized in the materials list before the procedure begins. Many are common stock solutions, others are commonly used buffers or media, while others are solutions unique to a particular protocol. Recipes for the latter solutions are provided in each unit, following the protocols (and before the commentary) under the heading Reagents and Solutions. It is important to note that the names of some of these special solutions might be similar from unit to unit (e.g., SDS sample buffer) while the recipes differ; thus, make certain that reagents are prepared from the proper recipes. On the other hand, recipes for commonly used stock solutions and buffers are provided once in APPENDIX Unavailable. These universal recipes are cross-referenced parenthetically in the materials lists rather than repeated with every usage. Throughout the manual, we have recommended commercial suppliers of chemicals, biological materials, and equipment. In some cases, the noted brand has been found to be of superior quality or it is the only suitable product available in the marketplace. In other cases, the experience of the author of that protocol is limited to that brand. In the latter situation, recommendations are offered as an aid to the novice protein science experimenter in obtaining the tools of the trade. Experienced investigators are therefore encouraged to experiment with substituting their own favorite brands. Addresses, phone numbers, and facsimile numbers of all suppliers mentioned in this manual are provided in the SUPPLIERS APPENDIX. Anyone carrying out these protocols may encounter the following hazardous or potentially hazardous materials: (1) radioactive substances, (2) toxic chemicals and carcinogenic or teratogenic reagents, and (3) pathogenic and infectious biological agents. Readers are referred to APPENDIX Unavailable and APPENDIX Unavailable for guidelines regarding use, storage, and disposal of these materials. Although cautionary statements are included in the appropriate units with cross-references to this appendix, we emphasize that users must proceed with the prudence and precaution associated with good laboratory practice, and that all materials must be used in strict accordance with local and national regulations. Most of the protocols included in this manual are used routinely in our own laboratories. These protocols work for us; to make them work for you we have annotated critical steps and included critical parameters and troubleshooting guides in the commentaries to most units. However, the successful evolution of this manual depends upon readers' observations and suggestions. Consequently, a self-mailing reader-response survey can be found at the back of the manual (and is included with each supplement); we encourage readers to send in their comments. Feedback can also be submitted online from the Current Protocols homepage at http://www.currentprotocols.com using the “Contact Us” link. This manual is the product of dedicated efforts by many of our scientific colleagues who are acknowledged in each unit and by the hard work by the Current Protocols editorial staff at John Wiley and Sons. The publisher's commitment and continuing support for a protein science manual, particularly through the efforts of Brian Crawford and Paul Majchrzyk, was essential for realizing this ambitious project. We are extremely grateful for the critical contributions by Virginia Chanda (Series Editor) who kept the editors and the contributors on track and played a key role in bringing the entire project to completion. Other skilled members of the Current Protocols staff who contributed to the project include Janet Blair, Hazel Chan, Elizabeth Konkle, Alice Ro, and Kathy Wisch. The extensive copyediting required to produce an accurate protocols manual was ably handled by Rebecca Barr, Karen Hopkin, Arline Keithe, Kathy Morgan, Connie Parks, and Allen Ranz, and electronic illustrations were prepared by Gae Xavier. Finally, we would like to thank two people who played important roles in initiating this project. Sarah Greene initially conceived this project and had a critical role in developing it. John A. Smith made important contributions during the early planning phases of the project. Helpful advice and criticisms of the project during early planning stages were provided by Fred Ausubel, Charles S. Craik, Bruce A. Cummingham, Mark Hermodson, Tony E. Hugli, Robert Kingston, Albert Light, Paul Matsudaira, David Moore, George Rose, and Kevin Struhl. Mark Hermodson read and critiqued the entire manual and individual chapters were thoughtfully reviewed by John Freed and John Kay. Finally, we are especially grateful to our many colleagues, who contributed material to this manual, for sharing their procedures and experiences. Easy-to-read overview of basic structural principles of proteins with extensive illustrations. Well established and clear review of protein structure and function, with an emphasis on enzymes. Deals with specific problems encountered when purifying membrane-associated proteins. Extensive treatment of 2-D gel analysis and its role in proteomics, including preparations of samples from diverse sources, alternative 2-D gel analysis methods and data analysis/interpretation. An updated look at protein structure in the genomics age. Includes discussion of sequence-to-structure and structure-to-function correlations. Overview of most current proteomics methods and supporting technologies. An updated biochemistry textbook whose protein-related chapters provide a comprehensive background in biochemistry.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W4245598151