Handbook of Endocrine Research Techniques

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ISBN: 9780122099205 | 0122099206
Cover: Hardcover
Copyright: 10/22/1993

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Written by experts in the field, Handbook of Endocrine Research Techniques is currently the only single source of up-to-date methods and strategies particularly useful in endocrinological research. As a resource for both the novice and experienced investigator, the book includes chapters which provide an introduction to the area, general concepts, detailed protocols, and extensive references.

Contributors	p. xv
Foreword	p. xix
Preface	p. xxiii
Hormone Assays
Radioimmunoassay and Radioreceptor Assay: Past, Present, and Future
History and Development of Radioimmunoassay	p. 4
Principles of Radioimmunoassay	p. 5
Principles of Immunoradiometric Assays	p. 10
Application of Antibody Fractionation in RIA	p. 15
Radioreceptor Assays	p. 16
Conclusions and Future Perspectives	p. 17
Appendix: Mathematical Aspects of Radioligand Assays	p. 18
References	p. 21
Immunoradiometric Assays
Introduction	p. 26
Principles of Immunoradiometric Assays	p. 27
Solid-Phase Systems	p. 30
Selection of Antibodies	p. 31
Variations in Immunoradiometric Assays	p. 36
Development of an Immunoradiometric Assay	p. 39
Factors Influencing Immunoradiometric Assays	p. 49
Advantages and Disadvantages of Immunoradiometric Assays	p. 50
Development of Nonradioactive Assays	p. 51
Conclusions	p. 52
References	p. 53
Enzyme-Linked Immunosorbent Assays
Background	p. 55
Overview of the General Principles Involved with ELISA	p. 56
Details of the ELISA Technique	p. 58
Related Techniques	p. 63
References	p. 64
Development and Characterization of Polyclonal and Monoclonal Antibodies
Introduction	p. 67
Immunogen Preparation	p. 69
Antisera Development	p. 70
Monoclonal Antibody Development	p. 74
Monoclonal Antibody Production	p. 86
Purification of Antisera and Monoclonal Antibodies	p. 87
Characterization of Antisera and Monoclonal Antibodies	p. 88
References	p. 90
Isolation of Polypeptide Hormones: General Separation Methods and Some Applications for Thyroid Hormones
Introduction	p. 93
Purification Techniques and Strategy	p. 94
Improvement in HPLC Fractionation of Thyroxine-Containing Peptides by Prior Acell Ion-Exchange Column Chromatography	p. 99
Identification of T3- and T4-Containing Peptides by HPLC with Photodiode Array (PDA) Detectors	p. 101
References	p. 105
Reverse Hemolytic Plaque Assays: Applications to Endocrine Problems
Introduction	p. 107
Procedures Common to All Plaque Assays	p. 110
Standard Plaque Assay	p. 113
Sequential Plaque Assay	p. 118
Simultaneous Plaque Assay	p. 121
Other Variations and Applications of Plaque Assays	p. 121
Summary	p. 124
References	p. 125
Capillary Electrophoresis Coupled to Fluorescence Detection for the Determination of Multiple Neuropeptides
Introduction	p. 127
A Capillary Electrophoresis-Based Assay	p. 132
Comments and Future Perspectives	p. 141
References	p. 143
Immunoreactive Heterogeneity in Peptide Measurement
General Introduction	p. 145
Proglucagon-Derived Peptides	p. 146
Conclusion	p. 154
References	p. 155
Why Use a Flow Cytometer for Endocrine Cell Analysis?
Introduction	p. 157
Capabilities of a Flow Cytometer	p. 158
Identification of Pituitary Cells by Flow Cytometric Immunofluorescence	p. 159
Sorting of Pituitary Cells by Light-Scatter Properties	p. 169
Sorting of Pituitary Cells by Fluorescence	p. 174
Concluding Remarks	p. 178
References	p. 179
Identification and Characterization of Insulin-like Growth Factor-Binding Proteins
Introduction	p. 181
Detection	p. 184
Identification of IGFBPs	p. 193
Conclusions	p. 203
References	p. 203
Histological and in Situ Approaches
Localization of Peptides: Double Labeling Immunohistochemistry
Preparation of Tissues	p. 208
Single Labeling	p. 212
Double Labeling	p. 214
Triple Labeling	p. 223
References	p. 224
Electron Microscopic Immunocytochemical Approaches to the Localization of Ligands, Receptors, Transducers, and Transporters
Introduction	p. 227
Use of Nonimmunologic Electron-Dense Hormone Complexes to Localize Receptors and Ligands	p. 228
General Consideration for Successful Immunocytochemical Electron Microscopy	p. 230
Localization of Hormones, Receptors, Transducers, and Transporters	p. 237
Future Prospects for Electron Microscopy	p. 259
Summary and Conclusion	p. 260
References	p. 261
In Situ Hybridization Histochemistry
Applications	p. 266
Methodological Issues	p. 271
In Situ Hybridization Protocols	p. 277
References	p. 285
Techniques for Receptors and Signal Transductors
Plasma Membrane Isolation Strategies for Cell Surface Receptors: Application for the Insulin Receptors
Introduction	p. 289
Preparation of Plasma Membranes	p. 290
General Considerations for Radioreceptor Assays	p. 296
Binding of 125I-Insulin to Plasma Membranes	p. 297
References	p. 299
Analysis of Autophosphorylation and Substrate Phosphorylation by Receptor Tyrosine Kinases
Introduction	p. 301
Methods	p. 306
References	p. 317
Involvement of Protein Tyrosine Phosphatase Activity in the Regulation of Insulin's Signal Transduction
Introduction	p. 321
Regulation of Protein Tyrosine Phosphatase Activity	p. 322
Cellular Functions Mediated by Protein Tyrosine Phosphatases	p. 323
Protein Tyrosine Phoshatases as Mediators of Insulin Action	p. 323
Alterations in Insulin Receptor-Protein Tyrosine Phosphatase Activity in Insulin-Resistant States	p. 324
Protein Substrates for Protein Tyrosine Phosphatases	p. 325
Assay of Protein Tyrosine Phosphatase Activity	p. 325
References	p. 335
Techniques for Evaluating Protein Kinase C in Intact Cells
Measuring Activation of Protein Kinase C in Intact Cells	p. 340
Down-Regulation of Protein Kinase C	p. 352
References	p. 354
Identification and Quantification of G-Proteins
Introduction	p. 357
Receptor-Stimulated GTPase	p. 358
Guanine Nucleotide-Binding Assays	p. 358
Labeling of G-Proteins by Bacterial Toxins	p. 360
Immunoblotting with Sequence-Specific Antisera	p. 362
References	p. 363
Determination of Adenylyl Cyclase Catalytic Activity
Considerations for Establishing Reaction Conditions	p. 366
Radioactive Substrates: [[superscript 3]H]ATP vs [[alpha]-[superscript 32]P]ATP	p. 372
Stopping the Reaction	p. 374
Chromatographic Alternatives	p. 378
Data Analysis	p. 386
References	p. 388
Intracellular Mediators of Peptide Hormone Action: Glycosyl Phosphatidylinositol/Inositol Phosphoglycan System
Introduction	p. 391
Methods for the Purification and Characterization of the Glycosyl Phosphatidylinositol/Inositol Phosphoglycan System	p. 393
Biological Activity of Inositol Phosphoglycan	p. 400
References	p. 404
Free Calcium Measurements: Down to the Single-Cell Level
Introduction	p. 407
Single Cell vs Population to Measure Ca[superscript 2+]	p. 408
Properties of Aequorin	p. 408
The Acquorin Technique	p. 410
Examples of Cytosolic Ca[superscript 2+] Measurements in Single Cells Using Aequorin	p. 415
References	p. 417
Molecular Techniques and Specific Model Systems
Approaches for the Purification, Quantitation, and Analysis of Hormone and Receptor mRNAs
General Introduction	p. 421
RNA Extraction	p. 422
Northern Blot Hybridization Analysis	p. 432
Solution Hybridization/RNase Protection Assay	p. 441
Primer Extension	p. 449
References	p. 454
Polymerase Chain Reaction: Applications to Endocrine Research
Introduction	p. 457
Polymerase Chain Reaction: Theory of the Method	p. 459
General Protocol: The Polymerase Chain Reaction	p. 462
Comments: General Polymerase Chain Reaction Protocol	p. 463
Reverse Transcription-Polymerase Chain Reaction	p. 466
Protocol for Reverse Transcription	p. 467
Comments: Reverse Transcription	p. 468
Quantitative Reverse Transcription-Polymerase Chain Reaction	p. 469
Contamination and Polymerase Chain Reaction False Positives	p. 469
RNA Template-Specific Polymerase Chain Reaction	p. 472
Subcloning Polymerase Chain Reaction Products	p. 474
Direct Sequencing of Polymerase Chain Reaction Products	p. 478
Some Commonly Encountered Polymerase Chain Reaction Artifacts	p. 480
Conclusions	p. 481
References	p. 482
Detection of Mutations in Hormone Receptor Genes
Introduction	p. 487
Southern Blotting	p. 488
Sequencing of Polymerase Chain Reaction-Amplified Genomic DNA and cDNA	p. 492
Allele-Specific Oligonucleotide Hybridization of Polymerase Chain Reaction Products	p. 495
Denaturing Gradient Gel Electrophoresis	p. 497
Single-Stranded Conformation Polymorphism (SSCP)	p. 500
References	p. 503
Techniques to Study Regulation of Gene Expression
RNA Analysis	p. 505
Direct Measurement of Transcriptional Rates	p. 506
Indirect Measurement of Transcriptional Rates	p. 508
References	p. 514
Transgenic Animals as a Tool in Endocrinology: Structure/Function Studies of Peptide Hormones Employing Transgenic Mice
Introduction	p. 515
Plasmid Construction and Mutagenesis	p. 517
Transgenic Mouse Production	p. 518
IGF-I Radioimmunoassay	p. 522
Immunoblotting Analysis	p. 522
Radioreceptor-Binding Assay	p. 524
Comments	p. 525
References	p. 526
Use of Nonmammalian Animal Models in Endocrine Investigation
Introduction	p. 530
Vertebrate Models for Hypothalamic Pituitary Interaction and Behavior	p. 534
Vertebrate Reproduction	p. 537
Vertebrate Hormones and Metabolism	p. 541
Invertebrate Models	p. 546
References	p. 549
Proliferation Induced by Growth Factors in Mammalian Fetal Cells
Introduction	p. 555
Isolation and Plating of Cells	p. 556
Establishment of Cell Quiescence	p. 557
Induction of Cell Proliferation	p. 560
References	p. 564
Expression of Hormones/Growth Factors and Their Receptors in Early Embryogenesis
Introduction	p. 567
The Mammalian Preimplantation Embryo	p. 569
The Chicken Embryo	p. 580
References	p. 586
Index	p. 589
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Handbook of Endocrine Research Techniques

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