Handbook of endocrine research techniques / edited by Flora de Pablo, Colin G. Scanes ; consulting editor, Bruce D. Weintraub.

Format:

Book

Contributor:

De Pablo, Flora.

Scanes, C. G.

Weintraub, Bruce D.

Language:

English

Subjects (All):

Hormones--Analysis.

Hormones.

Hormone receptors--Analysis.

Hormone receptors.

Molecular endocrinology--Technique.

Molecular endocrinology.

Endocrinology--methods.

Research Design.

Technique.

Medical Subjects:

Endocrinology--methods.

Research Design.

Physical Description:

xxiv, 599 pages : illustrations ; 24 cm

Other Title:

Endocrine research techniques.

Place of Publication:

San Diego : Academic Press, [1993]

Summary:

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.

Contents:

Part I Hormone Assays

1 Radioimmunoassay and Radioreceptor Assay: Past, Present, and Future / Sonia M. Najjar, Bruce D. Weintraub

I. History and Development of Radioimmunoassay 4

II. Principles of Radioimmunoassay 5

III. Principles of Immunoradiometric Assays 10

IV. Application of Antibody Fractionation in RIA 15

V. Radioreceptor Assays 16

VI. Conclusions and Future Perspectives 17

VII. Appendix: Mathematical Aspects of Radioligand Assays 18

2 Immunoradiometric Assays / M.R. Pandian, D.A. Fisher

II. Principles of Immunoradiometric Assays 27

III. Solid-Phase Systems 30

IV. Selection of Antibodies 31

V. Variations in Immunoradiometric Assays 36

VI. Development of an Immunoradiometric Assay 39

VII. Factors Influencing Immunoradiometric Assays 49

VIII. Advantages and Disadvantages of Immunoradiometric Assays 50

IX. Development of Nonradioactive Assays 51

3 Enzyme-Linked Immunosorbent Assays / Endre V. Nagy, Kenneth D. Burman

II. Overview of the General Principles Involved with ELISA 56

III. Details of the ELISA Technique 58

IV. Related Techniques 63

4 Development and Characterization of Polyclonal and Monoclonal Antibodies / Joan Rener

II. Immunogen Preparation 69

III. Antisera Development 70

IV. Monoclonal Antibody Development 74

V. Monoclonal Antibody Production 86

VI. Purification of Antisera and Monoclonal Antibodies 87

VII. Characterization of Antisera and Monoclonal Antibodies 88

5 Isolation of Polypeptide Hormones: General Separation Methods and Some Applications for Thyroid Hormones / Jorge Alemany, Jorge Garcia de Ancos, Enrique Mendez

II. Purification Techniques and Strategy 94

III. Improvement in HPLC Fractionation of Thyroxine-Containing Peptides by Prior Acell Ion-Exchange Column Chromatography 99

IV. Identification of T3- and T4-Containing Peptides by HPLC with Photodiode Array (PDA) Detectors 101

6 Reverse Hemolytic Plaque Assays: Applications to Endocrine Problems / L. Stephen Frawley

II. Procedures Common to All Plaque Assays 110

III. Standard Plaque Assay 113

IV. Sequential Plaque Assay 118

V. Simultaneous Plaque Assay 121

VI. Other Variations and Applications of Plaque Assays 121

7 Capillary Electrophoresis Coupled to Fluorescence Detection for the Determination of Multiple Neuropeptides / Juan P. Advis, Khurshid Iqbal, A. Waseem Malick, Norberto A. Guzman

II. A Capillary Electrophoresis-Based Assay 132

III. Comments and Future Perspectives 141

8 Immunoreactive Heterogeneity in Peptide Measurement / I. Valverde, M.L. Villanueva-Penacarrillo

II. Proglucagon-Derived Peptides 146

9 Why Use a Flow Cytometer for Endocrine Cell Analysis? / Frank M. Perez, Daniel R. Deaver, Wesley C. Hymer

II. Capabilities of a Flow Cytometer 158

III. Identification of Pituitary Cells by Flow Cytometric Immunofluorescence 159

IV. Sorting of Pituitary Cells by Light-Scatter Properties 169

V. Sorting of Pituitary Cells by Fluorescence 174

VI. Concluding Remarks 178

10 Identification and Characterization of Insulin-like Growth Factor-Binding Proteins / Yvonne W-H. Yang, Matthew M. Rechier

II. Detection 184

III. Identification of IGFBPs 193

Part II Histological and in Situ Approaches

11 Localization of Peptides: Double Labeling Immunohistochemistry / Jose E. Garcia-Arraras

I. Preparation of Tissues 208

II. Single Labeling 212

III. Double Labeling 214

IV. Triple Labeling 223

12 Electron Microscopic Immunocytochemical Approaches to the Localization of Ligands, Receptors, Transducers, and Transporters / Robert M. Smith, Leonard Jarett

II. Use of Nonimmunologic Electron-Dense Hormone Complexes to Localize Receptors and Ligands 228

III. General Consideration for Successful Immunocytochemical Electron Microscopy 230

IV. Localization of Hormones, Receptors, Transducers, and Transporters 237

V. Future Prospects for Electron Microscopy 259

13 In Situ Hybridization Histochemistry / Carolyn A. Bondy, Jian Zhou, Wei-Hua Lee

I. Applications 266

II. Methodological Issues 271

III. In Situ Hybridization Protocols 277

Part III Techniques for Receptors and Signal Transductors

14 Plasma Membrane Isolation Strategies for Cell Surface Receptors: Application for the Insulin Receptors / Maxine A. Lesniak, Joshua Shemer, Phillip Gorden

II. Preparation of Plasma Membranes 290

III. General Considerations for Radioreceptor Assays 296

IV. Binding of 125I-Insulin to Plasma Membranes 297

15 Analysis of Autophosphorylation and Substrate Phosphorylation by Receptor Tyrosine Kinases / Robert S. Garofalo

II. Methods 306

16 Involvement of Protein Tyrosine Phosphatase Activity in the Regulation of Insulin's Signal Transduction / Dalit Hecht, Yehiel Zick

II. Regulation of Protein Tyrosine Phosphatase Activity 322

III. Cellular Functions Mediated by Protein Tyrosine Phosphatases 323

IV. Protein Tyrosine Phoshatases as Mediators of Insulin Action 323

V. Alterations in Insulin Receptor-Protein Tyrosine Phosphatase Activity in Insulin-Resistant States 324

VI. Protein Substrates for Protein Tyrosine Phosphatases 325

VII. Assay of Protein Tyrosine Phosphatase Activity 325

17 Techniques for Evaluating Protein Kinase C in Intact Cells / Perry J. Blackshear

I. Measuring Activation of Protein Kinase C in Intact Cells 340

II. Down-Regulation of Protein Kinase C 352

18 Identification and Quantification of G-Proteins / Ravi lyenga

II. Receptor-Stimulated GTPase 358

III. Guanine Nucleotide-Binding Assays 358

IV. Labeling of G-Proteins by Bacterial Toxins 360

V. Immunoblotting with Sequence-Specific Antisera 362

19 Determination of Adenylyl Cyclase Catalytic Activity / Roger A. Johnson, Yoram Salomon

I. Considerations for Establishing Reaction Conditions 366

II. Radioactive Substrates: [[superscript 3]H]ATP vs [[alpha]-[superscript 32]P]ATP 372

III. Stopping the Reaction 374

IV. Chromatographic Alternatives 378

V. Data Analysis 386

20 Intracellular Mediators of Peptide Hormone Action: Glycosyl Phosphatidylinositol/Inositol Phosphoglycan System / Isabel Varela-Nieto, Luis Alvarez, Jose M. Mato

II. Methods for the Purification and Characterization of the Glycosyl Phosphatidylinositol/Inositol Phosphoglycan System 393

III. Biological Activity of Inositol Phosphoglycan 400

21 Free Calcium Measurements: Down to the Single-Cell Level / Antonio Sanchez-Bueno, Peter H. Cobbold

II. Single Cell vs Population to Measure Ca[superscript 2+] 408

III. Properties of Aequorin 408

IV. The Acquorin Technique 410

V. Examples of Cytosolic Ca[superscript 2+] Measurements in Single Cells Using Aequorin 415

Part IV Molecular Techniques and Specific Model Systems

22 Approaches for the Purification, Quantitation, and Analysis of Hormone and Receptor mRNAs / Martin L. Adamo, Bethel Stannard, Derek LeRoith, Charles T. Roberts, Jr.

II. RNA Extraction 422

III. Northern Blot Hybridization Analysis 432

IV. Solution Hybridization/RNase Protection Assay 441

V. Primer Extension 449

23 Polymerase Chain Reaction: Applications to Endocrine Research / Alan R.

Shuldiner, Riccardo Perfetti

II. Polymerase Chain Reaction: Theory of the Method 459

III. General Protocol: The Polymerase Chain Reaction 462

IV. Comments: General Polymerase Chain Reaction Protocol 463

V. Reverse Transcription-Polymerase Chain Reaction 466

VI. Protocol for Reverse Transcription 467

VII. Comments: Reverse Transcription 468

VIII. Quantitative Reverse Transcription-Polymerase Chain Reaction 469

IX. Contamination and Polymerase Chain Reaction False Positives 469

X. RNA Template-Specific Polymerase Chain Reaction 472

XI. Subcloning Polymerase Chain Reaction Products 474

XII. Direct Sequencing of Polymerase Chain Reaction Products 478

XIII. Some Commonly Encountered Polymerase Chain Reaction Artifacts 480

24 Detection of Mutations in Hormone Receptor Genes / Domenico Accili, Fabrizio Barbetti, Takashi Kadowaki, Simeon I. Taylor

II. Southern Blotting 488

III. Sequencing of Polymerase Chain Reaction-Amplified Genomic DNA and cDNA 492

IV. Allele-Specific Oligonucleotide Hybridization of Polymerase Chain Reaction Products 495

V. Denaturing Gradient Gel Electrophoresis 497

VI. Single-Stranded Conformation Polymorphism (SSCP) 500

25 Techniques to Study Regulation of Gene Expression / Fredric E. Wondisford

I. RNA Analysis 505

II. Direct Measurement of Transcriptional Rates 506

III. Indirect Measurement of Transcriptional Rates 508

26 Transgenic Animals as a Tool in Endocrinology: Structure/Function Studies of Peptide Hormones Employing Transgenic Mice / John J. Kopchick, Wen Y. Chen

II. Plasmid Construction and Mutagenesis 517

III. Transgenic Mouse Production 518

IV. IGF-I Radioimmunoassay 522

V. Immunoblotting Analysis 522

VI. Radioreceptor-Binding Assay 524

27 Use of Nonmammalian Animal Models in Endocrine Investigation / Ian P. Callard, Lynn Riddiford, Colin G. Scanes

II. Vertebrate Models for Hypothalamic Pituitary Interaction and Behavior 534

III. Vertebrate Reproduction 537

IV. Vertebrate Hormones and Metabolism 541

V. Invertebrate Models 546

28 Proliferation Induced by Growth Factors in Mammalian Fetal Cells / Manuel Benito, Margarita Lorenzo

II. Isolation and Plating of Cells 556

III. Establishment of Cell Quiescence 557

IV. Induction of Cell Proliferation 560

29 Expression of Hormones/Growth Factors and Their Receptors in Early Embryogenesis / Flora de Pablo, Gilbert A. Schultz, Susan Heyner

II. The Mammalian Preimplantation Embryo 569

III. The Chicken Embryo 580.

Notes:

Includes bibliographical references and index.

ISBN:

0122099206

OCLC:

27854470