Single molecule tools. Part A, Fluorescence based approaches / edited by Nils G. Walter.

Format:

Book

Contributor:

Walter, Nils G.

Series:

Methods in enzymology ; 472.

Methods in enzymology ; 472

Language:

English

Subjects (All):

Molecular biology--Methodology.

Molecular biology.

Enzymology.

Ultrastructure (Biology).

Physical Description:

1 online resource (559 p.)

Place of Publication:

London : Academic, 2010.

Language Note:

English

Summary:

Single molecule tools have begun to revolutionize the molecular sciences, from biophysics to chemistry to cell biology. They hold the promise to be able to directly observe previously unseen molecular heterogeneities, quantitatively dissect complex reaction kinetics, ultimately miniaturize enzyme assays, image components of spatially distributed samples, probe the mechanical properties of single molecules in their native environment, and ""just look at the thing"" as anticipated by the visionary Richard Feynman already half a century ago. This volume captures a snapshot of this vibrant, rap

Contents:

Front Cover; Methods in Enzymology; Copyright Page; Contents; Contributors; Preface; Volumes in Series; Chapter 1: Star Polymer Surface Passivation for Single-Molecule Detection; 1. Introduction; 2. Surface Grafting of PEO and Protein Repellence; 3. The NCO-sP(EO-stat-PO) System; 4. Preparation of sP(EO-stat-PO)-Coated Substrates for Single-Molecule Experiments; 5. Analysis of Protein Structure and Function on NCO-sP(EO-stat-PO) Surfaces; Acknowledgments; References

Chapter 2: Azide-Specific Labeling of Biomolecules by Staudinger-Bertozzi Ligation: Phosphine Derivatives of Fluorescent Probes Suitable for Single-Molecule Fluorescence Spectroscopy1. Introduction; 2. Materials and Methods; 2.1. Materials; 2.2. General methods; 2.3. Synthesis of Alexa488-phosphine (Fig. 2.1); 2.4. Synthesis of Cy3B-phosphine (Fig. 2.2); 2.5. Synthesis of Alexa647-phosphine20 Å (Fig. 2.3A); 2.6. Synthesis of Alexa647-phosphine24 Å (Fig. 2.3B); 2.7. Azide-specific labeling; 2.8. Quantitation of labeling efficiency; 2.9. Quantitation of labeling specificity; Acknowledgments

ReferencesChapter 3: Preparation of Fluorescent Pre-mRNA Substrates for an smFRET Study of Pre-mRNA Splicing in Yeast; 1. Introduction; 2. Identification of a Yeast Pre-mRNA with a Small Intron that is Spliced Efficiently In Vitro; 3. Synthetic Fluorescent Ubc4 Pre-mRNA; 4. Do the Dyes Affect the Efficiency of Splicing?; 5. Mutant Pre-mRNAs; 6. Tethering the Pre-mRNA to the Microscope Slide; 7. Summary and Conclusion; Acknowledgments; References; Chapter 4: Nanovesicle Trapping for Studying Weak Protein Interactions by Single-Molecule FRET; 1. Introduction; 2. Nanovesicle Trapping Approach

2.1. Lipid selection2.2. Lipid nanovesicle preparation and protein trapping; 3. smFRET Measurements of Weak Protein-Protein Interactions; 3.1. Surface immobilization of nanovesicles; 3.2. Control experiments; 3.3. Application to weak interactions between intracellular copper transporters; 4. Single-Molecule Kinetic Analysis of Three-State Protein-Protein Interactions; 5. Further Developments; 6. Concluding Remarks; Acknowledgments; References; Chapter 5: Droplet Confinement and Fluorescence Measurement of Single Molecules; 1. Introduction; 2. Methods for Droplet Generation

2.1. Emulsification2.2. Injection; 2.3. Microfluidics; 3. Methods for Droplet Manipulation; 3.1. Optical manipulation; 3.2. Lab-on-chip methods for droplet manipulation; 4. Droplet Coalescence and Mixing; 5. Experimental Considerations for Single Fluorophore Detection; 5.1. Protocol for aligning the apparatus; 5.2. Protocol for preparation of emulsion samples; 5.3. Protocol for droplet injection; 6. Single-Molecule Measurements in Droplets; 7. Future Prospects; Acknowledgments; References; Chapter 6: Single-Molecule Fluorescence Spectroscopy Using Phospholipid Bilayer Nanodiscs

1. Introduction

Notes:

Description based upon print version of record.

Includes bibliographical references and indexes.

ISBN:

9786612769887

9781282769885

128276988X

9780080959276

008095927X

OCLC:

701704000