Green Fluorescent Proteins (eBook)

Steve A. Kay, Kevin F. Sullivan (Herausgeber)

eBook Download: PDF

1998 | 1. Auflage
386 Seiten
Elsevier Science (Verlag)
978-0-08-085956-9 (ISBN)

Infos

This volume is an authoritative and comprehensive treatment of the approaches and techniques used for Green Fluorescent Proteins (GFP). The primary focus of this work is on research using biological systems. The volume covers all aspects of GFP, from its expression in different organisms to specific microscopic and data analysis methods.

Key Features
* Only volume on Green Fluorescent Protein research
* Covers all aspects of GFP
* Provides specific microscopic and data analysis methods
* Discusses the design and construction of GFP fusion proteins
* Covers GFP expression in animals, insects, plants, and microbes
* Details procedures for time lapse imaging of living cells
* Explains how to implement single molecule fluorescence detection with GFP
* Discusses dual label GFP strategies for multicolor fluorescence
* Presents fluorescence resonance energy transfer methods with GFPs
* Details quantitative fluorescence imaging techniques
* Extensively illustrated with color photographs

Inhalt

Front Cover 1
Methods in Cell Biology 4
Copyright Page 5
Contents 6
Contributors 12
Preface 16
Chapter I. Biophysics of the Green Fluorescent Protein 18
I. Introduction 18
II. Protein Folding and the Generation of This Chromophore 20
III. The Biophysics of the Fluorescence of GTP 25
IV. Resonance Energy Transfer Involving GFP 32
V. Summary 33
References 34
Chapter 2. Understanding Structure-Function Relationships in the Aequorea Victoria Green Fluorescent Protein 36
I. Introduction 36
II. Structure 37
III. Chromophore Formation 38
IV. Effects of Mutations on the Spectroscopic Properties of GFP 39
V. Effects of Mutations That Improve Thermosensitivity 43
VI. The Development of Enhanced Mutants 44
References 46
Chapter 3. Quantitative Imaging of the Green Fluorescent Protein (GFP) 48
I. Introduction 48
II. Factors That Influence/Limit Quantitation of GFP in Fluorescence Microscopy 49
III. Applications of LSCM for Quantitative Imaging of GFP 59
IV. Preparation of Purified GFP Samples 63
References 64
Chapter 4. Single-Molecule Fluorescence Detection of Green Fluorescence Protein and Application to Single-Protein Dynamics 66
I. Introduction 66
II. Design Considerations for Fluorescence Microscopes for Single-Molecule Detection 68
III. Characteristics of the Fluorescence from Single GFP Molecules 72
IV. Advantages of Using GFP for Single-Molecule Detection 81
V. GFP in Vitro and in Vivo Assays 82
Appendix I. Details of the TIR Microscope 84
Appendix II. Data Acquisition and Analysis 88
References 89
Chapter 5. Targeting GFP to Organelles 92
I. Introduction 92
II. Construction and Expression of the Organelle-Targeted GFP Chimeras 93
III. Dynamic Monitoring of Organelle Structure with the Targeted GFPs 96
IV. Expression in Primary Cultures 97
V. Visualizing GFP Chimeras with Different Spectral Properties 98
VI. Protocols 99
References 102
Chapter 6. Cytoskeletal Dynamics in Yeast 104
I. Introduction 104
II. Generating GFP Fusions 105
III. Imaging Considerations for Yeast Cells 110
IV. Time-Lapse Microscopy 111
V. Results: Cytoskeletal GFP Fusion Proteins 113
VI. Future 120
References 121
Chapter 7. Analysis of Nuclear Transport in Vivo 124
I. Introduction 124
II. Experimental Approaches and Protocols 125
References 138
Chapter 8. GFP Fusion Proteins as Probes for Cytology in Fission Yeast 140
I. Introduction 140
II. Expressing GFP Fusion Proteins 141
III. Applications of Fusion Proteins 148
References 154
Chapter 9. GFP Variants for Multispectral Imaging of Living Cells 156
1. Introduction 156
II. Green Fluorescent Protein Markers 157
III. Imaging of Living Cells 160
IV. Marking Different Cell Types in Arabidopsis 163
V. Spectrally Distinct Fluorescent Proteins for Multichannel Confocal Microscopy 164
VI. Summary 166
References 167
Chapter 10. GFP Fusions to a Microtubule Motor Protein to Visualize Meiotic and Mitotic Spindle Dynamics in Drosophila 170
I. Introduction 170
II. Labeling Strategies 171
III. Imaging GFP 173
IV. Applications of Ncd-GFP Imaging 176
V. Perspectives 179
References 179
Chapter 11. GFP as a Cell and Developmental Marker i n the Drosophila Nervous System 182
I. Introduction 182
II. Targeted Expression of GFP in Drosophila 185
III. Lines for Expression of GFP 185
IV. Visualizing GFP Expression 192
References 197
Chapter 12. Using Time-Lapse Confocal Microscopy for Analysis of Centromere Dynamics in Human Cells 200
I. Introduction 200
II. GFP Fusion Proteins 201
III. Microscopy 202
IV. Analysis 207
V. Summary 211
Appendix: Handling Confocal Images on the Laboratory Computer 212
References 217
Chapter 13. Visualization of Largescale Chromatin Structure and Dynamics Using the lac Operator/lac Repressor Reporter System 220
I. Introduction 220
II. Overview of Methodology 221
III. Construction of the lac Operator Repeat 223
IV. Manipulation of the lac Operator Repeats 225
V. Repressor-NLS and GFP-Repressor-NLS Constructs 227
VI. Gene Amplification and Cell Cloning 229
VII. Repressor Staining and Immunodetection of the lac Operator Repeat 231
VIII. In Vivo Observation of GFP-Repressor Localization 232
IX. Phototoxicity Issues 233
X. Present Results and Future Directions 235
References 237
Chapter 14. Centrosome Dynamics in Living Cells 240
I. Introduction 241
II. Cloning and Expression of GFP-Pericentrin 242
III. High-speed Microscopy 245
IV. Image Restoration by an Improved Deconvolution Method 248
V. Imaging Centrosomes 251
VI. Postimaging Confirmation of Centrosome Integrity and Function 252
References 255
Chapter 15. Transfections of Primary Muscle Cell Cultures with Plasmids Coding for GFP Linked to Full-Length and Truncated Muscle Proteins 256
I. Introduction 257
II. Construction of GFP-Linked Muscle Proteins 258
III. Preparation of Embryonic Avian Cardiomyocytes and Skeletal Muscle Myoblasts 260
IV. Methods of Transfection of Cross-Striated Cells in Culture 260
V. Transfection of Cross-Striated Muscle Cells with Full-Length cDNA for Sarcomeric Proteins 264
VI. Microscopic Observations of Live Cells 272
VII. Postprocessing of Transfected Cells 274
VIII. Problems Encountered in Cells Transfected with GFP-Sarcomeric Proteins 274
IX. Overview 275
References 275
Chapter 16. Monitoring the Dynamics and Mobility of Membrane Proteins Tagged with Green Fluorescent Protein 278
I. Introduction 279
II. Constructing and Expressing GFP Fusion Proteins: Strategies for Optimizing Brightness and Assessing Chimera Function 279
III. Practical Guidelines for the Preparation and Imaging of GFP-Expressing Cells 281
IV. Time-Lapse Imaging of GFP Chimeras: Critical Parameters 282
V. Analysis of Time-Lapse Imaging Data 285
VI. Relating GFP Chimera Fluorescence to Actual Numbers of GFP Molecules 287
VII. Fluorescence Recovery after Photobleaching FRAP 288
VIII. Qualitative FRAP Experiments 289
IX. Quantitative FRAP 292
X. Calculating D 293
XI. Fluorescence Loss in Photobleaching (FLIP) Using a Confocal Microscope 295
XII. Other Applications of Photobleaching 297
References 297
Chapter 17. Synchronous Real-Time Reporting of Multiple Cellular Events 300
I. Introduction 300
II. Green Fluorescent Protein 301
III. Luciferase 302
IV. Instrumentation and Techniques 303
V. Future Directions 307
References 308
Chapter 18. Visualizing Protein Interactions in Living Cells Using Digitized GFP Imaging and FRET Microscopy 310
I. Introduction 311
II. The Theory of FRET 312
III. Review of the FRET Literature 314
IV. Why Use FRET Microscopy? 315
V. Why Use the GFPs for FRET? 315
VI. Some Considerations for the Use of GFPs in FRET Imaging 318
VII. Some Considerations for Designing a FRET Imaging System 320
VIII. The Practical Application of FRET to Visualize Protein- Protein Interactions 324
IX. Overview and Conclusion 326
References 327
Chapter 19. Flow Cytometric Analysis and FACS Sorting of Cells Based on GFP Accumulation 332
I. General Introduction 332
II. Methods and Specific Applications 336
III. Typical Results 345
IV. Discussion and Conclusions 352
References 355
Chapter 20. GFP Biofluorescence: Imaging Gene Expression and Protein Dynamics in Living Cells 360
I. Introduction 361
II. Facilities 362
III. Maintaining Cells 364
IV. Imaging System 366
V. Computer Systems 372
VI. output 376
VII. Conclusions 382
References 383
Index 406

Produkt-Details

• Erscheint lt. Verlag: 7.10.1998
• Mitarbeit: Herausgeber (Serie): Paul T. Matsudaira, Leslie Wilson
• Sprache: englisch
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Zellbiologie
• Themenwelt: Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
• Themenwelt: Technik
• ISBN-10: 0-08-085956-9 / 0080859569
• ISBN-13: 978-0-08-085956-9 / 9780080859569