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
Contributors xi(4) Preface xv
1. Biophysics of the Green Fluorescent Protein 1(18) F.G. Prendergast I. Introduction 1(2) II. Protein Folding and the Generation of This Chromophore 3(5) III. The Biophysics of the Fluorescence of GTP 8(7) IV. Resonance Energy Transfer Involving GFP 15(1) V. Summary 16(1) References 17(2)
2. Understanding Structure-Function Relationships in the Aequorea victoria Green Fluorescent Protein 19(12) Andrew B. Cubitt Leslie A. Woollenweber Roger Heim I. Introduction 19(1) II. Structure 20(1) III. Chromophore Formation 21(1) IV. Effects of Mutations on the Spectroscopic Properties of GFP 22(4) V. Effects of Mutations That Improve Thermosensitivity 26(1) VI. The Development of Enhanced Mutants 27(2) References 29(2)
3. Quantitative Imaging of the Green Fluorescent Protein (GFP) 31(18) David W. Piston George H. Patterson Susan M. Knobel I. Introduction 31(1) II. Factors That Influence/Limit Quantitation of GFP in Fluorescence Microscopy 32(10) III. Applications of LSCM for Quantitative Imaging of GFP 42(4) IV. Preparation of Purified GFP Samples 46(1) References 47(2)
4. Single-Molecule Fluorescence Detection of Green Fluorescence Protein and Application to Single-Protein Dynamics 49(26) Daniel W. Pierce Ronald D. Vale I. Introduction 49(2) II. Design Considerations for Fluorescence Microscopes for Single-Molecule Detection 51(4) III. Characteristics of the Fluorescence from Single GFP Molecules 55(9) IV. Advantages of Using GFP for Single-Molecule Detection 64(1) V. GFP in Vitro and in Vivo Assays 65(2) Appendix I. Details of the TIR Microscope 67(4) Appendix II. Data Acquisition and Analysis 71(1) References 72(3)
5. Targeting GFP to Organelles 75(12) Francesca De Giorgi Zimran Ahmed Carlo Bastianutto Marisa Brini Laurence Sophie Jouaville Robert Marsault Marta Murgia Paolo Pinton Tullio Pozzan Rosario Rizzuto I. Introduction 75(1) II. Construction and Expression of the Organelle-Targeted GFP Chimeras 76(3) III. Dynamic Monitoring of Organelle Structure with the Targeted GFPs 79(1) IV. Expression in Primary Cultures 80(1) V. Visualizing GFP Chimeras with Different Spectral Properties 81(1) VI. Protocols 82(3) References 85(2)
6. Cytoskeletal Dynamics in Yeast 87(20) Janet L. Carminati Tim Stearns I. Introduction 87(1) II. Generating GFP Fusions 88(5) III. Imaging Considerations for Yeast Cells 93(1) IV. Time-Lapse Microscopy 94(2) V. Results: Cytoskeletal GFP Fusion Proteins 96(7) VI. Future 103(1) References 104(3)
7. Analysis of Nuclear Transport in Vivo 107(16) Paul Ferrigno Pamela A. Silver I. Introduction 107(1) II. Experimental Approaches and Protocols 108(13) References 121(2)
8. GFP Fusion Proteins as Probes for Cytology in Fission Yeast 123(16) Kenneth E. Sawin I. Introduction 123(1) II. Expressing GFP Fusion Proteins 124(7) III. Applications of Fusion Proteins 131(6) References 137(2)
9. GFP Variants for Multispectral Imaging of Living Cells 139(14) James Haseloff I. Introduction 139(1) II. Green Fluorescent Protein Markers 140(3) III. Imaging of Living Cells 143(3) IV. Marking Different Cell Types in Arabidopsis 146(1) V. Spectrally Distinct Fluorescent Proteins for Multichannel Confocal Microscopy 147(2) VI. Summary 149(1) References 150(3)
10. GFP Fusions to a Microtubule Motor Protein to Visualize Meiotic and Mitotic Spindle Dynamics in Drosophila 153(12) Sharyn A. Endow I. Introduction 153(1) II. Labeling Strategies 154(2) III. Imaging GFP 156(3) IV. Applications of Ncd-GFP Imaging 159(3) V. Perspectives 162(1) References 162(3)
11. GFP as a Cell and Developmental Marker in the Drosophila Nervous System 165(18) Andrea Brand I. Introduction 165(3) II. Targeted Expression of GFP in Drosophila 168(1) III. Lines for Expression of GFP 168(7) IV. Visualizing GFP Expression 175(5) References 180(3)
12. Using Time-Lapse Confocal Microscopy for Analysis of Centromere Dynamics in Human Cells 183(20) Kevin F. Sullivan Richard D. Shelby I. Introduction 183(1) II. GFP Fusion Proteins 184(1) III. Microscopy 185(5) IV. Analysis 190(4) V. Summary 194(1) Appendix: Handling Confocal Images on the Laboratory Computer 195(5) References 200(3)
13. Visualization of Large-Scale Chromatin Structure and Dynamics Using the lac Operator/lac Repressor Reporter System 203(21) Andrew S. Belmont Gang Li Gail Sudlow Carmen Robinett I. Introduction 203(1) II. Overview of Methodology 204(2) III. Construction of the lac Operator Repeat 206(2) IV. Manipulation of the lac Operator Repeats 208(2) V. Repressor-NLS and GFP-Repressor-NLS Constructs 210(2) VI. Gene Amplification and Cell Cloning 212(2) VII. Repressor Staining and Immunodetection of the lac Operator Repeat 214(1) VIII. In Vivo Observation of GFP-Repressor Localization 215(1) IX. Phototoxicity Issues 216(2) X. Present Results and Future Directions 218(2) References 220(4)
14. Centrosome Dynamics in Living Cells 224(16) Aaron Young Richard Tuft Walter Carrington Stephen J. Doxsey I. Introduction 224(1) II. Cloning and Expression of GFP-Pericentrin 225(3) III. High-Speed Microscopy 228(3) IV. Image Restoration by an Improved Deconvolution Method 231(3) V. Imaging Centrosomes 234(1) VI. Postimaging Confirmation of Centrosome Integrity and Function 235(3) References 238(2)
15. Transfections of Primary Muscle Cell Cultures with Plasmids Coding for GFP Linked to Full-Length and Truncated Muscle Proteins 240(21) Guissou A. Dabiri Kenan K. Turnacioglu Joseph C. Ayoob Jean M. Sanger Joseph W. Sanger I. Introduction 240(1) II. Construction of GFP-Linked Muscle Proteins 241(2) III. Preparation of Embryonic Avian Cardiomyocytes and Skeletal Muscle Myoblasts 243(1) IV. Methods of Transfection of Cross-Striated Cells in Culture 244(3) V. Transfection of Cross-Striated Muscle Cells with Full-Length cDNA for Sarcomeric Proteins 247(8) VI. Microscopic Observations of Live Cells 255(2) VII. Postprocessing of Transfected Cells 257(1) VIII. Problems Encountered in Cells Transfected with GFP-Sarcomeric Proteins 257(1) IX. Overview 258(1) References 258(3)
16. Monitoring the Dynamics and Mobility of Membrane Proteins Tagged with Green Fluorescent Protein 261(22) J. Lippincott-Schwartz J.F. Presley K.J.M. Zaal K. Hirschberg C.D. Miller J. Ellenberg I. Introduction 261(1) II. Constructing and Expressing GFP Fusion Proteins: Strategies for Optimizing Brightness and Assessing Chimera Function 262(2) III. Practical Guidelines for the Preparation and Imaging of GFP-Expressing Cells 264(1) IV. Time-Lapse Imaging of GFP Chimeras: Critical Parameters 265(3) V. Analysis of Time-Lapse Imaging Data 268(2) VI. Relating GFP Chimera Fluorescence to Actual Numbers of GFP Molecules 270(1) VII. Fluorescence Recovery after Photobleaching FRAP 271(1) VIII. Qualitative FRAP Experiments 272(3) IX. Quantitative FRAP 275(1) X. Calculating D 276(2) XI. Fluorescence Loss in Photobleaching (FLIP) Using a Confocal Microscope 278(2) XII. Other Applications of Photobleaching 280(1) References 280(3)
17. Synchronous Real-Time Reporting of Multiple Cellular Events 283(11) Jeffrey D. Plautz Steve A. Kay I. Introduction 283(1) II. Green Fluorescent Protein 284(1) III. Luciferase 285(1) IV. Instrumentation and Techniques 286(4) V. Future Directions 290(1) References 291(3)
18. Visualizing Protein Interactions in Living Cells Using Digitized GFP Imaging and FRET Microscopy 294(21) Ammasi Periasamy Richard N. Day I. Introduction 294(1) II. The Theory of FRET 295(2) III. Review of the FRET Literature 297(1) IV. Why Use FRET Microscopy? 298(1) V. Why Use the GEPs for FRET? 298(3) VI. Some Considerations for the Use of GFPs in FRET Imaging 301(2) VII. Some Considerations for Designing a FRET Imaging System 303(4) VIII. The Practical Application of FRET to Visualize Protein-Protein Interactions 307(2) IX. Overview and Conclusion 309(1) References 310(5)
19. Flow Cytometric Analysis and FACS Sorting of Cells Based on GFP Accumulation 315(29) David W. Galbraith Michael T. Anderson Leonard A. Herzenberg I. General Introduction 315(4) II. Methods and Specific Applications 319(9) III. Typical Results 328(7) IV. Discussion and Conclusions 335(3) References 338(6)
20. GFP Biofluorescence: Imaging Gene Expression and Protein Dynamics in Living Cells 344(25) Paul C. Goodwin I. Introduction 344(1) II. Facilities 345(2) III. Maintaining Cells 347(2) IV. Imaging Systems 349(6) V. Computer Systems 355(4) VI. Output 359(6) VII. Conclusions 365(1) References 366(3) Index 369
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