Probes and Tags to Study Biomolecular Function: for Proteins, RNA, and Membranes

In order to study living cells and their component molecules, two basic strategies have emerged in recent years. The first is to apply a soluble marker molecule, a ′probe′, that partitions to a particular cellular domain, and which can subsequently be measured. The second is to chemically or genetically modify a molecule of interest with a built–in probe, which is then called a ′tag′.
This handy reference systematically reviews current experimental methods and enables researchers to select the best solution for their experimental problems. For each method covered, the book provides step–by–step protocols, illustrated by typical research applications. Commencing with probing the lipid bilayer, the text then moves on to discuss probing proteins –– including membrane proteins –– and nucleic acids. This is the first single publication to incorporate chemical markers, fluorescent probes and genetic tags to allow a well–informed comparison of different solutions for similar problems in molecular analytics.

Spis treści:
Preface.
List of Contributors.
1. Fluorescent Sterols for the Study of Cholesterol Trafficking in Living Cells (Avery L. McIntosh, Huan Huang, Barbara P. Atshaves, Stephan M. Storey, Adalberto M. Gallegos, Thomas A. Spencer, Robert Bittman, Yoshiko Ohno–Iwashita, Ann B. Kier and Friedhelm Schroeder).
1.1 Introduction.
1.2 Methods for Imaging Fluorescent Sterols in Living Cells: Confocal and Multiphoton Laser Scanning Microscopy.
1.3 Cholesterol Structure and Distribution in Membranes.
1.4 NBD–Cholesterol.
1.5 Dansyl–Cholesterol.
1.6 BODIPY–Cholesterol.
1.7 Dehydroergosterol (DHE).
1.8 22–(p–Benzoylphenoxy)–23,24–bisnorcholan–5–en–3β–ol (FCBP) Photoactivatable Sterol.
1–9 BCÓ¨.
1.10 Conclusion.
References.
2. Li pid Binding Proteins to Study Localization of Phosphoinositides (Guillaume Halet and Patricia Viard).
2.1 Introduction: Phosphoinostitide Signaling.
2.2 Monitoring PI Distribution and Dynamics.
2.3 Detection of PtdIns (3,4,5)P2 Dynamics in Mouse Oocytes.
2.4 Monitoring PtdIns(4,5)P3 Synthesis after Stimulation with EGF.
2.5 Limitations of the Technique.
2.6 Conclusion.
References.
3. The Use of Lipid–Binding Toxins to Study the Distribution and Dynamics of Sphingolipids and Cholesterol (Reiko Ishitsuka and Toshihide Kobayashi).
3.1 Introduction.
3.2 Cholera Toxin.
3.3 Lysenin.
3.4 Perfringolysin O.
3.5 Aerolysin.
4. "FLAsH" Protein Labeling (Stefan Jakobs, Martin Andresen, and Christian A. Wurm).
4.1 Introduction.
4.2 Use of the Biarsenical–Tetracysteine System in S. cerevisae.
4.3 Short Protocols.
4.4 Troubleshooting.
References.
5. AGT/SNAP–Tag: A Versatile Tag for Covalent Protein Labeling (Amaud Gautier, Kai Johnsson and Helen Oâ Hare).
5.1 Introduction.
5.2 Labeling SNAP–Tag Fusion Proteins with BG Derivatives.
5.3 SNAP–Tag for Cell Imaging.
5.4 Procedures for SNAP–Tag Labeling.
5.5 Broader Applications of SNAP–Tag to Study Protein Function.
5.6 Conclusion.
References.
6. Trimethoprim Derivatives for Labeling Dihydrofolate Reductase Fusion Proteins in Living Mammalian Cells (Lawrence W. Miller and Virginia W. Cornish).
6.1 Introduction.
6.2 Preparation of E. coli Expression Vectors.
6.3 Synthesis of Fluorescent Trimethoprim Derivatives.
6.4 Cell Growth and Transfection.
6.5 Protein Labeling and Microscopy.
6.6 Results and Discussion.
6.7 Conclusion.
References.
7. Phosphopantetheinyl Transferase Catalyzed Protein Labeling and Molecular Imaging (Norman J. Marshall and Jun Yin).
7.1 Introduction.
7.2 Protein Posttranslationa l Modification by Phosphopantetheinyl Transferases.
7.3 Protein Labeling Via Carrier Protein Fusions.
7.4 Orthogonal Protein Labeling by Short Peptide Tags.
7.5 Summary and Perspectives.
References.
8. Bioorthogonal Chemical Transformations in Proteins by an Expanded Genetic Code (Brigit Wiltschi and Nediljkp Budisa)
8.1 Introduction.
8.2 Chemical Transformations at the Protein N–terminus Classical Approaches.
8.3 Chemical Transformations Using an Expanded Genetic Code.
8.4 Modifications Internal to Proteins.
8.5 Chemical Transformations at the Protein C–terminus.
8.6 Summary and Outlook.
References.
9. Using the Bacteriophage MS2 Coat Protein–RNA Binding Interaction to Visualize RNA in Living Cells (Jeffrey A. Chao, Kevin Czaplinski and Robert H. Singer).
9.1 Introduction.
9.2 Construction of an MBS–Containing Reporter RNA.
9.3 Construction of an MS2 CP–FP Chimera.
9.4 Co–introduction of MS2 Reporter RNA and MS2 CP–FP.
9.5 Microscopy Platform for Single Molecule Detection of RNAs in Living Cells.
9.6 Protocols for Co–expressing MS2 CP–FP–and MBS–Containing Plasmids for Live Cell Imaging.
9.7 Image Analysis and Quantification of mRNA Molecules.
References.
Index.

Nota biograficzna:
Lawrence Miller is Assistant Professor of Chemistry at the University of Illinois at Chicago. He obtained both his B.S. and Ph.D. from the University of Wisconsin–Madison. He then moved to Columbia University in the City of New York to initiate an independent research project specialising in novel imaging technology for the biosciences. His research interests include chemical methods to advance molecular imaging, in vivo study of biochemical and biomolecular function and bioanalytical chemistry. He has 3 patents and several papers to his name.

Okładka tylna:
In order to study living cells and their component molecules, two basic strategies have emerged in recent years. The first is to apply a soluble marker molecule, a ′probe′, that partitions to a particular cellular domain, and which can subsequently be measured. The second is to chemically or genetically modify a molecule of interest with a built–in probe, which is then called a ′tag′.
This handy reference systematically reviews current experimental methods and enables researchers to select the best solution for their experimental problems. For each method covered, the book provides step–by–step protocols, illustrated by typical research applications. Commencing with probing the lipid bilayer, the text then moves on to discuss probing proteins –– including membrane proteins –– and nucleic acids. This is the first single publication to incorporate chemical markers, fluorescent probes and genetic tags to allow a well–informed comparison of different solutions for similar problems in molecular analytics.