Green Fluorescent Proteins

Cover
Kevin F. Sullivan, Steve A. Kay, Paul T. Matsudaira
Gulf Professional Publishing, 1999 - 386 Seiten
The green fluorescent Protein (GFP) has initiated a revolution in molecular cell biology by establishing a general approach for visualizing nearly any protein of interest in any cell, tissue or species. Researchers working at all levels of biology, such as single molecule dynamics, protein trafficking within cells, organelle dynamics and cell and tissue behaviors during development, have made important use of GFP.
This volume is a practical guide to working with GFP and contains discussions of GFP biophysics and GFP mutants, design of GFP fusion proteins and expression systems, and detailed protocols for introducing and visualizing GFP in living cells. Methods for quantitative imaging, data analysis and presentation are detailed by a select group of researchers who have made some of the key contributions to bio-fluorescent imaging technologies.
Key Features
* 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 flourescence imagine techniques
* Extensively illustrated with color photographs
 

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Inhalt

Monitoring the Dynamics and Mobility of Membrane Proteins Tagged
16
Andrew B Cubitt Leslie A Woollenweber and Roger Heim
19
Quantitative Imaging of the Green Fluorescent Protein GFP
31
Applications of LSCM for Quantitative Imaging of GFP
42
with Green Fluorescent Protein
47
SingleMolecule Fluorescence Detection of Green Fluorescence Protein
49
Targeting GFP to Organelles
75
Cytoskeletal Dynamics in Yeast
87
Transfection of CrossStriated Muscle Cells with FullLength cDNA for Sarcomeric Proteins
247
Microscopic Observations of Live Cells
255
Postprocessing of Transfected Cells
257
Overview
258
J LippincottSchwartz J F Presley K J M Zaal K Hirschberg C D Miller and J Ellenberg I Introduction
261
Brightness and Assessing Chimera Function
262
Practical Guidelines for the Preparation and Imaging of GFPExpressing Cells
264
Critical Parameters
265

Analysis of Nuclear Transport in Vivo
107
GFP Fusion Proteins as Probes for Cytology in Fission Yeast
123
Applications of Fusion Proteins
131
GFP Fusions to a Microtubule Motor Protein to Visualize Meiotic
153
GFP as a Cell and Developmental Marker in the Drosophila
165
Visualizing GFP Expression
175
Using TimeLapse Confocal Microscopy for Analysis of Centromere
183
Visualization of LargeScale Chromatin Structure and Dynamics Using the lac Operatorlac Repressor Reporter System Andrew S Belmont Gang Li Gai...
203
Overview of Methodology
204
Construction of the lac Operator Repeat
206
Manipulation of the lac Operator Repeats
208
RepressorNLS and GFPRepressorNLS Constructs
210
Gene Amplification and Cell Cloning
212
Repressor Staining and Immunodetection of the lac Operator Repeat
214
In Vivo Observation of GFPRepressor Localization
215
Phototoxicity Issues
216
Present Results and Future Directions
218
References
220
Centrosome Dynamics in Living Cells Aaron Young Richard Tuft Walter Carrington and Stephen J Doxsey
223
Introduction
224
Cloning and Expression of GFPPericentrin
225
HighSpeed Microscopy
228
Image Restoration by an Improved Deconvolution Method
231
Imaging Centrosomes
234
Postimaging Confirmation of Centrosome Integrity and Function
235
References
238
Transfections of Primary Muscle Cell Cultures with Plasmids Coding for GFP Linked to FullLength and Truncated Muscle Proteins Guissou A Dabiri ...
239
Introduction
240
Construction of GFPLinked Muscle Proteins
241
Preparation of Embryonic Avian Cardiomyocytes and Skeletal Muscle Myoblasts
243
Methods of Transfection of CrossStriated Cells in Culture
244
Analysis of TimeLapse Imaging Data
268
Relating GFP Chimera Fluorescence to Actual Numbers of GFP Molecules
270
Fluorescence Recovery after Photobleaching FRAP
271
Qualitative FRAP Experiments
272
Quantitative FRAP
275
Calculating D
276
Fluorescence Loss in Photobleaching FLIP Using a Confocal Microscope
278
Other Applications of Photobleaching
280
Synchronous RealTime Reporting of Multiple Cellular Events Jeffrey D Plautz and Steve A Kay I Introduction
283
Green Fluorescent Protein
284
Luciferase
285
Instrumentation and Techniques
286
Future Directions
290
References
291
Visualizing Protein Interactions in Living Cells Using Digitized GFP Imaging and FRET Microscopy
293
Ammasi Periasamy and Richard N Day I Introduction
294
The Theory of FRET
295
Review of the FRET Literature
297
Why Use FRET Microscopy?
298
Some Considerations for the Use of GFPs in FRET Imaging
301
Some Considerations for Designing a FRET Imaging System
303
The Practical Application of FRET to Visualize Protein Protein Interactions
307
Overview and Conclusion
309
References
310
Imaging Gene Expression and Protein Dynamics
343
Imaging Systems
349
Output
359
Conclusions
365
Index
369
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Über den Autor (1999)

University of California, Santa Barbara, USA

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