Reverse Chemical Genetics Methods and Protocols

Now a major approach in the identification of certain gene product functions, reverse chemical genetics allows the identification of target genes to precede phenotypic changes created by specific small molecules. In Reverse Chemical Genetics: Methods and Protocols, expert authors introduce fundamental resources, including databases for reverse chemical genetics, as well as the modification of these cDNA resources by the most recent molecular technologies. The book explores newly established methods in addition to potentially applicable techniques that make use of specific small molecules, plus functional assays utilizing these molecules, while focusing on methods that make feasible the functional discovery and comprehensive knowledge of particular target genes. As a volume in the highly successful Methods in Molecular Biology™ series, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Reverse Chemical Genetics: Methods and Protocols promises to inspire researchers to conduct their own experiments in order to progress this vital field of study.

Part I: The Importance of Fully-Characterized Genomic Resources and Informatics for Reverse Chemical Genetics 1. ORFeome Cloning Osamu Ohara 2. Systematic Cloning of an ORFeome Using the Gateway System Akihisa Matsuyama and Minoru Yoshida 3. High-Throughput Construction of ORF Clones for Production of the Recombinant Proteins Hisashi Yamakawa 4. Computational Overview of GPCR Gene Universe to Support Reverse Chemical Genomics Study Makiko Suwa and Yukiteru Ono 5. High-Performance Gene Expression Module Analysis Tool and Its Application to Chemical Toxicity Data Wataru Fujibuchi, Hyeryung Kim, Yoshifumi Okada, Takeaki Taniguchi, and Hideko Sone 6. Human Protein Reference Database and Human Proteinpedia as Discovery Tools for Systems Biology T.S. Keshava Prasad, Kumaran Kandasamy, and Akhilesh Pandey Part II: Modification of cDNA Resources Utilized for Reverse Chemical Genetics 7. High-Throughput Production of the Recombinant Proteins Expressed in Escherichia coli Utilizing cDNA Resources Kiyo Shimada and Hisashi Koga 8. An Insect Cell-Free System for Recombinant Protein Expression Using cDNA Resources Takashi Suzuki, Toru Ezure, Masaaki Ito, Masamitsu Shikata, and Eiji Ando 9. Recombinant Protein Production by a Kaiko-Baculovirus System Hidekazu Nagaya 10. Pulse-Chase Experiment for the Analysis of Protein Stability in Cultured Mammalian Cells by Covalent Fluorescent Labeling of Fusion Proteins Kei Yamaguchi, Shinichi Inoue, Osamu Ohara, and Takahiro Nagase Part III: The Refinement of Established Methods by Recent Innovations 11. Application of 2D-DIGE in Cancer Proteomics toward Personalized Medicine Tadashi Kondo and Setsuo Hirohashi 12. Fully Automated Two-Dimensional Electrophoresis System for High-Throughput Protein Analysis Atsunori Hiratsuka and Kenji Yokoyama 13. Large-Scale Whole Mount In Situ Hybridization of Mouse Embryos Hirohito Shimizu, Kenta Uchibe, and Hiroshi Asahara 14. Identification of the Specific Binding Proteins of Bioactive Small Compound Using Affinity Resins Akito Tanaka Part IV: Potential Applicable Techniques for Reverse Chemical Genetics 15. Protein Microarrays: Effective Tools for the Study of Inflammatory Diseases Xiaobo Yu, Nicole Schneiderhan-Marra, Hsin-Yun Hsu, Jutta Bachmann, and Thomas O. Joos 16. Preparation of Highly Sensitive Protein Array Using Reactive Polymer Toshifumi Shiroya, Hiroyuki Tanaka, Minako Hanasaki, and Hisao Takeuchi 17. High-Throughput SPR Biosensor Motoki Kyo, Kimihiko Ohtsuka, Eiji Okamoto, and Kazuki Inamori 18. Drug Discovery through Functional Screening in the Drosophila Heart Takeshi Akasaka and Karen Ocorr 19. Permeable Cell Assay: A Method for High-Throughput Measurement of Cellular ATP Synthetic Activity Kiyotaka Y. Hara 20. Novel Methodology for Immobilization of Biomolecules on the Surface of a Photo-Responsible Polymer Containing Azobenzene Moiety Osamu Watanabe