Enzyme assays are among the most frequently performed procedures in biochemistry and are routinely used to estimate the amount of enzyme present in a cell or tissue, to follow the purification of an enzyme, or to determine the kinetic parameters of a system. The range of techniques used tomeasure the rate of an enzyme-catalysed reaction is limited only by the nature of the chemical change and the ingenuity of the investigator. This book describes the design and execution of enzyme assays, covering both general principles and specific chapters.Building upon the highly popular first edition, this book combines revised or rewritten chapters with entirely new contributions. Topics include experimental protocols covering photometric, radiometric, HPLC, and electrochemical assays, along with methods for determining enzyme assays after gelelectrophoresis. The theory underlying each method is outlined, together with a description of the instrumentation, sensitivity and sources of error. Also included are chapters on the principles of enzyme assay and kinetic studies; techniques for enzyme extraction; high- throughout screening;statistical analysis of enzyme kinetic data; and the determination of active site concentration.This second edition of Enzyme Assays will be valuable not only to biochemists, but to researchers in all areas of the life sciences.
Edited by one of the leading experts in the field, this book fills the need for a book presenting the most important methods for high-throughput screenings and functional characterization of enzymes. It adopts an interdisciplinary approach, making it indispensable for all those involved in this expanding field, and reflects the major advances made over the past few years. For biochemists, analytical, organic and catalytic chemists, and biotechnologists.
Enzyme Assays S. Gul, S.K. Sreedharan and K. Brocklehurst The development of an assay procedure by which the course of a particular enzyme-catalyzed reaction may be monitored is an essential preliminary step in virtually all experimental studies on the enzyme. This book summarizes the key principles underlying enzyme assays, including assay design and the evaluation of experimental data. It provides details of and references to the major physico-chemical techniques commonly used in enzymology. Useful ancillary data are also included on subjects such as active site titration, reagent kits and buffers. The Essential Data series provides rapid access to the core data required by researchers on a daily basis, in convenient pocket-sized volumes.
A practice-oriented guide to assaying more than 100 of the most important enzymes, complete with the theoretical background and specific protocols for immediate use in the biochemical laboratory. Now expanded with a new section on metal ion determination.
This book offers comprehensive information on all aspects of ELISA, starting with the fundamentals of the immune system. It also reviews the history of analytical assays prior to the advent of ELISA (enzyme-linked immunosorbent assay) and addresses the materials of choice for the fabrication of the platforms, possible biomolecular interactions, different protocols, and evaluation parameters. The book guides readers through the respective steps of the analytical assay, while also familiarizing them with the possible sources of error in the assay. It offers detailed insights into the immobilization techniques used for protein attachment, as well as methods for evaluating the assay and calculating the key parameters, such as sensitivity, specificity, accuracy and limit of detection. In addition, the book explores the advantages and shortcomings of the conventional ELISA, as well as various approaches to improving its performance. In this regard, merging and integrating other technologies with widely known ELISAs have opened new avenues for the advancement of this immunoassay. Accordingly, the book provides cutting-edge information on integrated platforms such as ELISpot, plasmonic ELISAs, sphere-/bead-based ELISAs, paper-/fiber-based ELISAs and ELISA in micro-devices.
Methods to Determine Enzymatic Activity is a textbook about industrial enzymes. The book features definitions, classifications and applications of selected enzymes important in industry and in biotechnological processes. Analytical methods for these enzymes are also included in the text. The main objective of this textbook is to provide readers information focused on the current analysis methods of enzymatic activity at qualitative and quantitative levels. Each chapter is about one specific enzyme and contains information about its substrate and some biochemical properties. The methodologies are presented as an experimental protocol allowing interested readers to reproduce the experimental methods detailed within the textbook. These protocols contain the principle of the technique, materials, methods, and all steps necessary for the determination of enzyme activity and interpretation of results. Each methodology is illustrated with photos and schemes for a better and clear understanding. This book, therefore, uniquely brings modern analysis techniques of industrial enzymes in a single easy to understand volume. This textbook is suitable for undergraduate enzymology courses and advanced industrial biotechnology and microbiology courses.
The use of High Performance Liquid Chromatography (HPLC) techniques in the study of enzymatic reactions has grown significantly since the publication of the first edition of this highly successful book: the role of enzymes in biological research has expanded; the application of HPLC and enzymes has extended to more disciplines; advances in separation techniques and instrumentation have increased the capability of HPLC; and the discovery of new enzymes has spawned new methods of analysis. High Performance Liquid Chromatography in Enzymatic Analysis, Second Edition addresses these developments in its coverage of the refinements of HPLC methods and their use in a wide range of laboratory applications. It offers the same practical approach found in the first edition, incorporates a wealth of new information into existing chapters, and adds new chapters to deal with new applications, including capillary electrophoresis, forensic chemistry, microdialysis, and the polymerase chain reaction. Topics include: * Application of HPLC to the assay of enzymatic activities * Concepts and principles of HPLC, including the latest technological advances * Concepts and principles of capillary electrophoresis (CE) * Strategy for design of an HPLC/CE system for assay of enzyme activity * Preparation of enzymatic activities from tissues and single cells * Analysis of enzymatic activities in body fluids, including chromatobiosis * HPLC for the identification of new enzymatic activities * Fundamentals of the polymerase chain reaction * HPLC in forensics * Survey of enzymatic activities assayed by the HPLC method, including many new categories * Multienzyme systems, including many new examples * HPLC in the analysis of contaminated food "It is the ability of HPLC to accomplish separations completely and rapidly that led to its original application to problems in the life sciences, particularly those related to purification. An analysis of the literature revealed that this technique was used primarily for the purification of small molecules, macromolecules such as peptides and proteins, and more recently, antibodies. This application to purification has all but dominated the use of the method, and there has been a plethora of books, symposia, and conferences on the use of HPLC for these purposes. However, it was only a matter of time before others began to look beyond and to explore the possibilities that result from the capacity to make separations quickly and efficiently." --from the preface to the First Edition Easy to read and full of practical advice and hundreds of diagrams and examples, High Performance Liquid Chromatography in Enzymatic Analysis, Second Edition is an invaluable resource for students, researchers, and laboratory workers in analytical chemistry and biochemistry, molecular biology and cell biology, and for anyone interested in keeping up with this fast-growing field.
Methods of Soil Enzymology provides the first comprehensive set of vetted methods for studying enzymes in soils. Readers will especially benefit from the step-by-step explanation of the lab procedures, as well as background information for using these methods effectively and analyzing data. Main topics include activity assays, enzyme extraction, and synthetic enzyme complexes. Each method covered includes background informaton, step-by-step descriptions of the procedure, and special comments regarding nuances, pitfalls, and interpretation of the method. Learn the latest research methods, including enzyme extraction methods and procedures for creating synthetic enzyme complexes, as well as the newest ways to use small-scale and high-throughput methods for enzyme activity assays. Written for the researcher, but welcoming to those new to soil enzymology, the introduction includes conceptual information to orient those who are not familiar with these methods but want to use them. In the tradition of SSSA methods books, Methods of Soil Enzymology features a comprehensive approach with a focus on ease of use.
Enzyme Activity in Single Cells, Volume 628, the latest release in the Methods of Enzymology series, discusses groundbreaking cellular physiology research that is taking place in the biological sciences. Chapters in this new release cover Spatial and temporal resolution of caspase waves in single Xenopus eggs during apoptosis, Spatial and temporal organization of metabolic complexes in cells, Measuring cellular efflux and biomolecular delivery: synthetic approaches to imaging and engineering cells, Slide-based, single-cell enzyme assays, Single-cell assays using integrated continuous-flow microfluidics, High-throughput screening of single-cell lysates, Microfluidic capture of single cells for drug resistance assays, and much more.
