Radioisotopes in Biology

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ISBN: 9780199638260 | 0199638268
Cover: Paperback
Copyright: 2/1/2002

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The book provides an introduction to the use of radioactivity in the bioscience laboratory. An introduction sets the scene, before two chapters that cover general aspects of radioactivity. Methods for the detection of radioactivity are covered in two chapters that deal with scintillationcounting and autoradiography. Four chapters are concerned with the radioisotope protocols used to study key cellular processes: radiolabelling of nucleic acids and proteins, subcellular localisation and identification of macromolecules, immunoassay methods and pharmacological techniques. Twochapters provide a summary of legislative requirements in the USA and European Union and there is an Appendix of useful facts and addresses.

Preface

Protocol list

Abbreviations

xix

Introduction

(6)

Robert J. Slater

The decision to use a radioisotope

(1)

Laboratory facilities

(1)

The choice of a labelled compound

(3)

The choice of a radionuclide

(1)

Labelling position

(1)

Specific activity

(1)

The isotope effect

(1)

Storage and purity

(1)

Experimental design

(2)

Radioisotope use

(24)

David Prime

Barry Frith

Introduction

(1)

Radioactivity

(7)

Radioactive decay

(3)

Units of radioactivity

(1)

Properties of radiations

(1)

Radiation dose and equivalent dose

(1)

External radiation doses from beta emitters

(1)

External radiation doses from X-rays and gamma rays

(1)

Internal radiation doses from radionuclides

(1)

Biological effects of radiation and the basis of legislation

(2)

Effective dose

(1)

Tissue level effects

(1)

Carcinogenic effects

(1)

Genetic effects

(1)

Biological basis of dose limits

(2)

Risk rates for biological effects

(2)

Working with unsealed sources

(2)

Getting started

(1)

Designation of working areas

(1)

Designation of `classified workers'

(1)

Controlling the risks to radiation workers

(1)

The non-classified workers

(1)

The classified worker

(1)

The tracer study

(3)

Experimental design

(2)

The purchase of isotopes and labelled compounds

(1)

Receiving the isotope

(1)

Isotope manipulations

(1)

Design of isotope laboratory

(1)

Personal protection in the radioisotope laboratory

(1)

Routine monitoring of area

(1)

External dose measurement

(1)

Measurement of air contamination

(1)

Measurement of surface contamination

(1)

Estimation of individual dose

(1)

Transport of radioactive material

(2)

Requirements and controls for transport of excepted packages

(1)

Disposal of radioactive waste

(3)

Solid waste

(1)

Liquid low-level waste (aqueous and approved liquid scintillation cocktails)

(1)

References

(1)

Risk assessments for the use of radioisotopes in biology

(32)

Peter E. Ballance

Introduction

(1)

The Health and Safety at Work etc. Act 1974

(1)

The Management of Health and Safety at Work Regulations 1999

(2)

The risk assessment process

(2)

The content of the risk assessment

(1)

Responsibility for making risk assessments

(1)

Suggested methods for making risk assessments

(9)

General laboratory hazards

(1)

Use of generic risk assessments in biological research and teaching

(8)

Hazards and risks to health from the use of radioactive substances and ionizing radiation

(2)

Special hazard and risk assessment for 125I

(2)

The hazard

(1)

Risks to health and radiation protection procedures to be followed

(2)

Elimination of the hazard

(1)

Special hazard and risk assessment for DNA labelling/sequencing work with 32P

(5)

Minimizing uptakes

(1)

Assessment of risks and control of external radiation from 32P

(4)

Risk assessments for gamma-emitting radionuclides

(2)

Risk assessments for environmental protection

(1)

Risks to the health of biological research workers in relation to other risks

(5)

References

(3)

Radioisotope detection using X-ray film

(22)

Ron A. Laskey

Introduction

(1)

Choosing a class of detection method: autoradiography versus fluorography or intensifying screens

(1)

Direct autoradiography gives high resolution but limited sensitivity

(1)

Fluorography increases sensitivity for weak β emitters

(1)

Intensifying screens increase sensitivity for high-energy β particles or γ rays

(1)

Direct autoradiography

(1)

Procedure for direct autoradiography

(1)

Use of X-ray intensifying screens to increase sensitivity for 32P and γ-ray emitters

(3)

Fluorography of weak β emitters

(9)

General technical procedures for fluorography of acrylamide gels by all methods

(1)

Procedures for fluorography of acrylamide gels

(4)

Fluorography of other samples excluding acrylamide gels

(3)

General technical considerations for radioisotope detection by X-ray film

(3)

Choice of film and screens

(1)

Conditions for exposure and processing of film

(1)

Pre-exposure of film to hypersensitize it for use with intensifying screens or fluorography

(2)

Procedures for drying acrylamide or agarose gels

(1)

The underlying principles of radioisotope detection by X-ray film

(3)

Acknowledgements

(1)

References

(2)

The scintillation counter

(46)

Charles L. Dodson

Introduction

(1)

Counting efficiency and counting error

(1)

Counting error

(1)

Origin and nature of nuclear emissions

(2)

Interaction of decay species with matter

(2)

The liquid scintillation counter

(16)

Introduction

(1)

The scintillation cocktail

(2)

Beta particle spectrum

(1)

The liquid scintillation counter

(12)

Advanced topics

107

(4)

Multi-label counting

107

(2)

Colour quench detection and correction

109

(1)

D.p.m. correction without quench curves

110

(1)

Sample monitoring

111

(1)

Special counting situations

111

(2)

Cerenkov counting

111

(2)

Low-level counting

113

(1)

Solid scintillators and programmable coincidence gates

113

(1)

Sample preparation

114

(6)

Cocktail properties

115

(1)

Sample types

116

(4)

Vials

120

(1)

Gamma counters

120

(4)

Gamma counter operational parameters

121

(3)

Flow counters

124

(1)

Multi-detector liquid scintillation and gamma counters

125

(1)

Storage phosphor technology

125

(6)

Phosphor screens

126

(1)

Scanning unit

126

(1)

Phosphor screen erasure

127

(1)

Quantitative analysis

128

(1)

References

128

(1)

Further reading

129

(2)

In vitro labelling of nucleic acids and proteins

131

(40)

Martin W. Cunningham

A. Patel

A. C. Simmonds

D. Williams

Introduction

131

(2)

Choice of radiolabel

131

(2)

Applications of nucleic acid labelling

133

(1)

Labelling methods for hybridization probes

134

(20)

Random prime labelling

136

(2)

Nick translation

138

(3)

Transcription labelling

141

(4)

End-labelling of oligonucleotides

145

(6)

Choosing a radioactive labelling system for nucleic acid probes

151

(3)

Cycle sequencing with 33P-labelled terminators

154

(2)

Applications of labelled proteins

156

(1)

Labelling of proteins with 125I

156

(8)

General considerations

156

(1)

Direct labelling

156

(5)

Indirect methods

161

(1)

Purification of radio-iodinated proteins

162

(1)

Determination of specific activity

163

(1)

Labelling of proteins with 3H and 14C

164

(1)

Tritium labelling

164

(1)

Carbon-14 labelling

165

(1)

Protein kinase assays

165

(3)

In vitro translation with radiolabelled amino acids

168

(3)

Acknowledgements

169

(1)

References

170

(1)

Subcellular localization of biological molecules

171

(38)

Richard Cumming

Ian Durrant

Rachel Fallon

Introduction

171

(1)

Compartmentalization within the cell

171

(1)

Subcellular localization of proteins

172

(9)

Introduction

172

(1)

Western blotting

173

(3)

Immunoprecipitation

176

(2)

Histological techniques

178

(3)

Subcellular localization of nucleic acids

181

(24)

Identification of nucleic acid molecules isolated from subcellular fractions

181

(1)

Localization of nucleic acids using ISH

182

(3)

Safety considerations when using ISH (32)

185

(1)

Preparation of target cells or tissues

186

(4)

Preparation of radiolabelled probe

190

(2)

Hybridization

192

(3)

Autoradiography

195

(4)

Analysis of data

199

(1)

Controls required for ISH

199

(6)

Simultaneous localization of proteins and nucleic acids

205

(1)

Conclusion

205

(4)

Acknowledgements

206

(1)

References

206

(3)

Radioisotopes and immunoassay

209

(24)

Adrian F. Bristow

Andrew J. H. Gearing

Robin Thorpe

Introduction

209

(1)

Immunoassays: theoretical considerations

210

(3)

Radioimmunoassays

213

(13)

Reagents

213

(6)

Assay conditions

219

(1)

Separation techniques

219

(2)

Specific protocols

221

(2)

Interpretation of results

223

(3)

Immunoradiometric assays

226

(3)

Choice of solid support

226

(1)

Antibodies for IRMAs

226

(1)

Radiolabelling antibodies

227

(1)

Assay design

227

(2)

Non-radioisotopic immunoassays

229

(4)

Enzyme immunoassays

229

(1)

Radioisotopes versus non-isotopic methods in immunoassay: future trends

230

(2)

References

232

(1)

Pharmacological techniques

233

(32)

Isabel J. M. Beresford

Michael J. Allen

Introduction

233

(1)

Radioligand binding assays

233

(13)

Introduction

233

(1)

Choice of reagents

234

(2)

Separation techniques

236

(4)

Validation and optimization of assay

240

(1)

Data analysis

241

(4)

Non-radioactive assays

245

(1)

G-protein-coupled receptor second messenger assays

246

(10)

Introduction

246

(1)

Measurement of [35S]GTPγS binding

247

(3)

Measurement of 3',5'-cyclic adenosine monophosphate (cAMP)

250

(3)

Measurement of inositol triphosphate

253

(2)

Non-radioactive assays

255

(1)

Enzyme assays

256

(5)

Introduction

256

(1)

Measurement of nitric oxide synthase activity using [14C]arginine

256

(5)

Homogeneous assays

261

(1)

Ion channel assays

261

(2)

Introduction

261

(1)

Measurement of Na+ channel function by [14C]guanidine flux

261

(2)

Some commercially available curve-fitting programs

263

(2)

Acknowledgements

263

(1)

References

263

(2)

Summary of legislation in radiological protection in the UK

265

(6)

David Prime

Barry Frith

Introduction

265

(1)

The Radioactive Substances Act (1993)

265

(1)

Acquisition

265

(1)

Storage

265

(1)

Radioactive waste

265

(1)

The Ionising Radiations Regulations (1999)

266

(5)

Interpretation and general

266

(1)

General principles and procedures

266

(2)

Arrangements for the management of radiation protection

268

(1)

Designated areas

268

(1)

Classification and monitoring of persons

268

(1)

Arrangements for the control of radioactive substances, articles and equipment

269

(1)

Duties of employees and miscellaneous

269

(2)

Federal regulations on use of radionuclides in the USA

271

(18)

Fileen D. Hotte

Neal Nelson

Introduction

271

(2)

Licensing requirements

273

(1)

General domestic license

273

(1)

Specific licenses of broad scope

274

(1)

Medical uses

275

(1)

Exemptions

276

(1)

Radiation Protection-Nuclear Regulatory Commission

276

(4)

Immediate telephone report

279

(1)

Twenty-four hour telephone report

279

(1)

Telephone report within 30 days

280

(1)

30 day written report

280

(1)

Other sections of Title 10, Code of Federal Regulations

280

(1)

Radiation protection-Department of Labor

280

(1)

Transportation

281

(1)

Department of Transportation

281

(1)

United States Postal Service

281

(1)

Environment-Environmental Protection Agency

282

(1)

Human uses-Department of Health and Human Services

282

(1)

Historical perspective and future developments

283

(4)

Conclusion

287

(2)

References

287

(2)

Appendices

289

(14)

A1 Properties and radiation protection data of isotopes commonly used in the biological sciences

290

(1)

A2 Types of monitor

291

(2)

A3 Curie-becquerel conversion chart

293

(1)

A4 Calculation of amount of radioactivity remaining (RR) after a given half-life (NL) has passed

294

(1)

A5 Units commonly used to describe radioactivity

295

(1)

A6 Licensing and advisory authorities

296

(1)

A7 List of suppliers

297

(6)

Index

303

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Radioisotopes in Biology

Summary

Table of Contents

Supplemental Materials