Adrenergic receptors are important modulators in the sympathetic c- trol of various metabolic processes in the central and peripheral nervous s- tems. These receptors are localized at multiple sites throughout the central nervous system (CNS) and serve as important regulators of CNS-mediated behavior and neural functions, including mood, memory, neuroendocrine c- trol, and stimulation of autonomic function. Adrenergic Receptor Protocols consists of 35 chapters dealing with va- ous aspects of adrenergic receptor analyses, including the use of genetic, RNA, protein expression, transactivator, second messenger, immunocytochemical, electrophysiological, transgenic, and in situ hybridization approaches. This volume details the use of various methods to examine the adrenergic receptor system, using aspects of the genetic flow of information as a guide (DNA? RNA ? transactivator ? protein expression ? second messenger analyses ? cellular analyses ? transgenic whole animal approaches). Adrenergic Receptor Protocols displays step-by-step methods for s- cessful replication of experimental procedures, and would be useful for both experienced investigators and newcomers in the field, including those beg- ning graduate study or undergoing postdoctoral training. The Notes section contained in each chapter provides valuable troubleshooting guides to help develop working protocols for your laboratory. With Adrenergic Receptor Protocols, it has been my intent to develop a comprehensive collection of modern molecular methods for analyzing adrenergic receptors. I would like to thank the many chapter authors for their contributions.
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978-1-59259-684-3 (9781592596843)
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Genetic Analysis.- Construction of Libraries for Isolation of Adrenergic Receptor Genes.- Isolation of Adrenergic Receptor Genes.- Analyses of Adrenergic Receptor Sequences.- Polymerase Chain Reaction Screening of Genomic Libraries for Adrenergic Receptor Genes.- Solution-Phase Library Screening for Identification of Rare Adrenergic Receptor Clones.- Genetic Polymorphisms of Adrenergic Receptors.- RNA Analysis.- Northern Blot Analyses Detecting Adrenergic Receptor mRNAs.- Ribonuclease Protection Assay for the Detection of ?1-Adrenergic Receptor RNA.- Determination of Adrenergic Receptor mRNAs by Quantitative Reverse Transcriptase-Polymerase Chain Reactions.- Nuclear Run-On Assays for Measurement of Adrenergic Receptor Transcription Rate.- Primer Extension Methods for Determination of ?1-Adrenergic Receptor mRNA Start Sites.- Expression Analysis.- Use of Eukaryotic Vectors for the Expression of Adrenergic Receptors.- Expression of ?-Adrenergic Receptors in Recombinant Baculovirus-Infected Insect Cells.- Expression of ?-Adrenergic Receptors in E. coli.- Use and Pharmacological Analysis of Established and Transfected Cell Lines Expressing Adrenergic Receptors.- Transient Transfection and Adrenergic Receptor Promoter Analysis.- Antisense RNA/DNA-Based Techniques to Probe Adrenergic Receptor Function.- Targeted Disruption of Adrenergic Receptor Genes.- Receptor and Second Messenger Analysis.- Development of Antibodies to Adrenergic Receptors.- Western Blot Detection of Adrenergic Receptors.- UV Crosslinking of Adrenergic Receptors and Ligands.- Detection of ?-Adrenergic Receptors by Radioligand Binding.- Assessing Adrenergic Receptor Conformation Using Chemically Reactive Fluorescent Probes.- Biochemical Methods for Detection and Measurement of Cyclic AMP and AdenylylCyclase Activity.- Assay of Arachidonic Acid Release Coupled to ?1- and ?2-Adrenergic Receptors.- Patch-Clamp Recording Methods for Examining Adrenergic Regulation of Potassium Currents in Ocular Epithelial Cells.- Transactivator Analysis.- Southwestern Blots for Detection of a DNA Binding Protein Recognizing the ?1B-Adrenergic Receptor Gene Promoter.- DNase I Footprinting Analysis of Transcription Factors Recognizing Adrenergic Receptor Gene Promoter Sequences.- Electrophoretic Mobility Shift Assay for Detection of DNA Binding Proteins Recognizing ?-Adrenergic Receptor Gene Sequences.- Determination of mRNA Stability and Characterization of Proteins Interacting with Adrenergic Receptor mRNAs.- Microscopy Analysis.- Use of Immunohistochemistry and Confocal Microscopy in the Detection of Adrenergic Receptors.- Distribution of ?2A- and ?2C-Adrenergic Receptor Immunoreactivity in the Central Nervous System.- Quantitative Light Microscopic Autoradiography of ?2-Adrenergic Radioligand Binding Sites.- In Situ Hybridization of Adrenergic Receptor mRNA in Brain.- Use of Electron Microscopy in the Detection of Adrenergic Receptors.
