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This book explores new protocols and features revised and improved methodologies, making it a valuable reference for all those contemplating a move into the study of intracellular Ca2+. It features lab-ready procedures and includes key implementation advice.
This volume looks at the latest developments in studying calcium signaling. The chapters in this book discuss topics such as in vitro and in vivo analyses in model systems; methods to measure Ca2+ in different subcellular compartments; single Ca2+ channels; methods to measure cellular ROS and ATP; and the functionality of the ATP synthase. 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 tips on troubleshooting and avoiding known pitfalls. Comprehensive and cutting-edge, Calcium Signalling: Methods and Protocols is a valuable resource that covers both conceptual and methodological viewpoints to aid beginners and experts in furthering their studies in the developing field of calcium homeostasis research.
This detailed volume explores techniques for performing calcium signaling in diverse cell types, alongside detailed methods for data analysis. Beginning with chapters on cellular assays to assess calcium signaling, the book continues with sections on imaging methods, methods to assess calcium within primary cilia, the endoplasmic reticulum, and the mitochondria, as well as tissue-specific calcium signaling methods, including the establishment of primary cell or tissue models and measuring calcium in whole organisms such as Drosophila. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Calcium Signaling: Methods and Protocols serves as an ideal guide for laboratories of all types seeking to measure calcium robustly.
In the first edition of Calcium Signaling Protocols I began by writing “The regula- 2+ tion of intracellular Ca is a common theme presented in many papers over the last 20 2+ or so years and the description of the Ca -sensitive indicator dye fura-2 in 1985 resulted in a massive increase in these types of studies. ” This statement is as true in 2005 as it was in 1999, but 20 or so years is now 30 years! There has been some reorganization of the volume such that there are now 22 ch- ters including five new ones, all written by experts in their field. These new chapters 2+ include use of the FlexStation and electrophysiological measurement of Ca channel activity. The book is broken into six parts. Part I is a general coverage of basic theory and the simplest use of fluorescent indicators. Part II covers specialist measurement 2+ systems and Part III covers measurement of Ca channel activity. Assessment of 2+ release of stored Ca is covered in some detail in Part IV, with Parts V and VI cover- 2+ ing specialist measurement techniques and Ca -sensitive targets. Putting a book like this together, even as a second edition, takes time and I am, again, indebted to the individual authors for their help and patience. I am also very grateful to Professor John M. Walker, the series editor, for his continued help and advice over the course of this project.
The book combines general concepts and methods to investigate calcium signalling in cells ranging from molecular biology approaches to manipulation of calcium in living cells. The focus within these methods in on the broad range of fluorescence imaging technology, in particular on optical sectioning techniques and fast image acquisition. In addition to these general guidelines there are application examples in a context beyond calcium signalling in two major fields: investigations of isolated cardiac myocytes and red blood cell related research. While the cellular cardiology section provides snapshots of certain calcium signalling aspects, the red blood cell part presents an overview from the functional identification of calcium-channels to a concept of physiological and pathophysiological relevance.
Arguably no other field of biological research embraces such a diverse array of experimental approaches as does the field of calcium signaling. Not only does it span virtually all conceptual and technical areas of molecular and cell biology, but a number of unique techniques, such as the use of permeabilized cells to study intracellular calcium metabolism, have evolved directly from calcium research. Calcium Signaling explores this fascinating area of research, covering the biophysical, molecular biological, and cell biological techniques involved in the study of calcium signaling. This work examines a broad range of topics-from techniques for imaging and measuring calcium in subcellular compartments, to subcellular fractionation and intracellular calcium stores, and patch clamp investigation of calcium channels. Filled with detailed illustrations, Calcium Signaling addresses those key methodological approaches that are useful to scientists and researchers investigating calcium function and regulation in many diverse areas of biological and medical research.
Calcium plays an important role in a wide variety of biological processes. This divalent metal ion can bind to a large number of proteins; by doing so it modifies their biological activity or their stability. Because of its distinct che- cal properties calcium is uniquely suited to act as an on–off switch or as a light dimmer of biological activities. The two books entitled Calcium-Binding Protein Protocols (Volumes I and II) focus on modern experimental analyses and methodologies for the study of calcium-binding proteins. Both extracel- lar and intracellular calcium-binding proteins are discussed in detail. H- ever, proteins involved in calcium handling (e. g. , calcium pumps and calcium channels), fall outside of the scope of these two volumes. Also, calcium-bi- ing proteins involved in bone deposition will not be discussed, as this specific topic has been addressed previously. The focus of these two books is on studies of the calcium-binding proteins and their behavior in vitro and in vivo. The primary emphasis is on protein chemistry and biophysical methods. Many of the methods described will also be applicable to proteins that do not bind calcium. Calcium-Binding Protein Protocols is divided into three main sections. The section entitled Introduction and Reviews provides information on the role of calcium in intracellular secondary messenger activation mechanisms. Mo- over, unique aspects of calcium chemistry and the utilization of calcium in dairy proteins, as well as calcium-binding proteins involved in blood clotting, are addressed.
Calcium plays an important role in a wide variety of biological processes. This divalent metal ion can bind to a large number of proteins; by doing so it modifies their biological activity or their stability. Because of its distinct che- cal properties calcium is uniquely suited to act as an on–off switch or as a light dimmer of biological activities. The two books entitled Calcium-Binding Protein Protocols (Volumes I and II) focus on modern experimental analyses and methodologies for the study of calcium-binding proteins. Both extracel- lar and intracellular calcium-binding proteins are discussed in detail. H- ever, proteins involved in calcium handling (e. g. , calcium pumps and calcium channels), fall outside of the scope of these two volumes. Also, calcium-bi- ing proteins involved in bone deposition will not be discussed, as this specific topic has been addressed previously. The focus of these two books is on studies of the calcium-binding proteins and their behavior in vitro and in vivo. The primary emphasis is on protein chemistry and biophysical methods. Many of the methods described will also be applicable to proteins that do not bind calcium. Calcium-Binding Protein Protocols is divided into three main sections. The section entitled Introduction and Reviews provides information on the role of calcium in intracellular secondary messenger activation mechanisms. Mo- over, unique aspects of calcium chemistry and the utilization of calcium in dairy proteins, as well as calcium-binding proteins involved in blood clotting, are addressed.
As our understanding of the biological sciences expands, the bou- aries between traditional disciplines tend to blur at the edges. Physio- gists and pharmacologists, for instance, now need to embrace techniques that until recently were the strict preserves of biochemists and mole- lar biologists. However, the acquisition of new technologies can be a time-consuming and frustrating business, and unless an expert is on hand to give instruction, precious hours can be spent poring over half-described Methods sections with no guarantee of eventual success. The aim of Signal Transduction Protocols has been to get experts with "hands-on" experience in particular techniques to give detailed accounts of experimental protocols in a recipe-type format, which we hope will circumvent the problems of ambiguity often encountered when reading the literature. The techniques described in Signal Transduction Protocols are those that we think will be most useful in addressing questions in the area of receptor-mediated cell signaling, with particular regard to those receptors that are part of the G-protein-linked superfamily. To keep it to a manageable size, we have omitted any reference to electrophysi- ogy and have instead concentrated on more biochemical approaches.