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Assay Development Fundamentals and Practices

by: Wu, Ge

Assay Development Fundamentals and Practices

by: Wu, Ge

ISBN 13:
9780470191156
ISBN 10:
0470191155
Edition: 1st
Format: Hardcover
Copyright: 04/26/2010
Publisher: Wiley

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Summary

The first comprehensive, integrated treatment of the subject, Assay Development: Fundamentals and Practices covers the essentials and techniques involved in carrying out an assay project in either a biotechnology/drug discovery setting or a platform setting. After discussing the basics, it explains the treatment of assays for purified protein, cell-based assays, and high-throughput screening. It includes good practices and case studies. This is a premier resource for biotechnologists, analytical chemists, biochemists, medicinal chemists, toxicologists, and others, and an excellent text for professors preparing students to enter the biotechnology and pharmaceutical industries.

Foreword	p. xi
Preface	p. xiii
Acknowledgments	p. xv
Abbreviations	p. xvii
Introduction To Assay Development	p. 1
Assay and Bioassay	p. 1
Drug Discovery Process and Role of Assays in the Process	p. 1
Bioassay Development	p. 17
Bioassay Classifications	p. 22
Useful Websites	p. 24
Bibliography	p. 24
Measurement And Instrumentation	p. 27
Measurement and Perturbation	p. 27
Common Instrumental Methods and Instrument Components	p. 32
Molecular Absorption Measurements	p. 39
Molecular Luminescence Measurements	p. 43
Luminescence Lifetime Measurement and Time-Resolved Fluorescence Measurement	p. 53
Fluorescence Resonance Energy Transfer (FRET) and Time-Resolved-FRET	p. 54
Fluorescence Quenching	p. 57
Fluorescence Polarization (FP)	p. 58
Radioactivity Measurement	p. 61
Evaluating and Selecting an Instrumental Method for Bioassay	p. 64
Useful Websites	p. 68
Bibliography	p. 68
Fundamental Principles of Assay Development With Isolated Proteins	p. 69
Chemical Potential, Equilibrium, and Kinetics	p. 70
Protein Binding Studies at Equilibrium	p. 72
Kinetic Studies of the Protein Binding Process	p. 77
Enzyme Kinetics	p. 83
Inhibition of Protein Function	p. 89
Assay Development with Isolated Proteins	p. 98
Useful Websites	p. 103
Bibliography	p. 104
Separation-Based Techniques in Bioassays	p. 105
Washing Solid Supports to Remove Impurities	p. 105
Organic Solvent Extraction of Hydrophobic Molecules	p. 107
Centrifugation to Remove Dense Particles	p. 108
Membrane Filtration	p. 109
Liquid Chromatography	p. 110
Electrophoresis	p. 117
Useful Websites	p. 122
Bibliography	p. 122
General Protein Binding Assay Formats	p. 125
Equilibrium Dialysis	p. 126
Competitive Binding Assays with Radioactive or Other Labeled Ligands	p. 128
Application of SPA and FlashPlate in Binding Studies	p. 131
Application of Fluorescence Polarization (FP) in Binding Studies	p. 132
Application of FRET Assays in Binding Studies	p. 137
Application of ELISA in Binding Studies	p. 139
Surface Plasmon Resonance (SPR) Technology and Its Application in Binding Studies	p. 143
Application of Label-Free Technologies in Binding Studies	p. 151
Useful Websites	p. 152
Bibliography	p. 153
Functional Assays With Isolated Proteases	p. 155
Introduction to Proteases and Their Substrates	p. 155
Function of Proteases and Their Role in Drug Discovery	p. 159
Protease Assays	p. 161
Protease Substrate Profiling	p. 166
Protease Inhibitors	p. 168
Assay Development for Caspases with a Fluorogenic Substrate	p. 170
Assay Development for Carboxypeptidase U (EC 3.4.17.20)	p. 173
Useful Websites for Proteases	p. 176
Bibliography	p. 177
Functional Assays For Protein Kinases	p. 181
Introduction to Protein Kinases	p. 181
Substrates for In Vitro Kinase Assays	p. 182
Kinase Assay Development Strategies	p. 186
Kinase Assay Based on Detection of Phosphorylated Product	p. 187
Kinase Assays by Measuring the Generation of ADP	p. 199
Kinase Assays by Measuring the Depletion of ATP	p. 200
Kinase Assays by Measuring the Depletion of Peptide Substrate	p. 204
Kinase Assays by Simultaneous Measurement of Both Product and Substrate	p. 204
Example of a Kinase Assay Development in HTRF Format	p. 205
Useful Websites	p. 209
Bibliography	p. 209
Fundamental Principles Of Cell-Based Assays	p. 213
Cell Signaling, Signal Transduction, and Cellular Responses	p. 214
General Approaches in Cell-Based Assays	p. 221
Concept of Affinity and Efficacy in Cell-Based Assays	p. 223
Development of Cell-Based Assays	p. 228
Useful Websites	p. 235
Bibliography	p. 235
Functional Ion Channel Assays	p. 239
Introduction to Ion Channels	p. 239
Strategies for Ion Channel Assays	p. 243
Electrophysiological Methods	p. 247
Ion Flux Methods	p. 253
Membrane Potential Sensing Methods	p. 259
Selecting Suitable Assays for Ion Channel Studies	p. 261
Useful Websites and Vendors	p. 262
Bibliography	p. 262
Assays With GPCRs	p. 265
Introduction to GPCRs and G Proteins	p. 265
G Protein-Coupled Receptor Activation and Signal Transduction	p. 267
Strategies of GPCR Assay Development	p. 269
G Protein-Coupled Receptor Assays by Measuring the Extent of GTP Binding to Ga	p. 270
G Protein-Coupled receptor Assays Based on Measurement of cAMP	p. 272
G Protein-Coupled Receptor Assays Based on Measurement of Intracellular Inositol Phospholipids	p. 276
G Protein-Coupled Receptor Assays Based on Measurement of Intracellular Ca²+	p. 277
G Protein-Coupled Receptor Assays Based on Measurement of MAPK Activity	p. 278
G Protein-Coupled Receptor Assays with Reporter Gene	p. 279
G Protein-Coupled Receptor Assays by Monitoring Events Leading to GPCR Internalization	p. 282
Useful Websites	p. 285
Bibliography	p. 285
Assays Based on Integrated Cell System Properties	p. 289
Cell Viability, Proliferation, and Cytotoxicity Assays	p. 289
Measurement of Extracellular Indicators of Cellular Metabolism	p. 296
Measurement of Cell's Effect on Electrical Impedance	p. 298
Measurement of Protein Secretion from Cells	p. 301
Measurement of Discoloration of Melanophore Cells	p. 302
Measurement of Cell Motility	p. 303
Useful Websites	p. 304
Bibliography	p. 304
High-Content Cell-Based Assay With Optical Imaging Techniques	p. 307
Sample Preparation	p. 308
Cellular Image Collection	p. 312
Image Abstraction, Analysis, and Data Management	p. 316
Applications of iCHCS	p. 318
Useful Websites	p. 319
Bibliography	p. 320
High-Throughput Screening	p. 321
Introduction	p. 321
Molecular or Cellular Targets and Assay Development	p. 325
Compound Library Management	p. 326
Hardware Module	p. 332
Software Module	p. 344
HTS Operation Management	p. 347
Building an HTS Operation for Biopharmaceutical Discovery	p. 355
Quality Control and Data Analysis in Primary Screening	p. 359
Useful Websites	p. 368
Bibliography?	p. 369
Case Study: Development Of A Microfluidic-Based Kinase Assay Platform	p. 371
Background of Microfluidic Technology and Its Application in Bioassays	p. 371
The Original Mobility Shift Kinase Assay Format	p. 377
Realizing the Flaws in the Original Kinase Assay Format	p. 384
Searching for Alternative Kinase Assay Methods	p. 398
Development of the Off-Chip Kinase Assay Format	p. 400
Current Stage of Microfluidic Technology in Bioassays	p. 411
Appendix: Poster Presented at the 2002 Society for Biomolecular Screening Annual Meeting: Analysis of Mobility Shift Data Obtained from Labchip in Kinase Assay	p. 413
Useful Websites	p. 421
Bibliography	p. 421
Index	p. 423
Table of Contents provided by Ingram. All Rights Reserved.

Amazon no longer offers textbook rentals. We do!

Assay Development Fundamentals and Practices

Assay Development Fundamentals and Practices

9780470191156

0470191155

Summary

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