Two-Hybrid Systems
Humana Press Inc. (Verlag)
978-0-89603-808-0 (ISBN)
Many, if not all, essential biological processes require selective interactions between proteins. Complex signaling systems require sequential, ordered protein-protein interactions at essentially all levels of the signaling cascade. For example, peptide hormones interact with selective membrane receptor proteins, and autophosphorylation of the receptor then recruits other key regulatory proteins that initiate kinase cascades in which each phosphorylation event requires selective recognition of the protein substrate. The ultimate signaling effect, in many cases, is the regulation of RNA polymerase II-directed transcr- tion in the nucleus, a process that involves numerous, multiprotein complexes important for transcription initiation, elongation, termination, and reinitiation. Defining, characterizing, and understanding the relevance of these protein- protein interactions is an arduous task, but substantial inroads have been made over the past 20 years.
The development of more recent methodologies, such as mammalian expression systems, immunopurification schemes, expression cloning strategies, surface plasmon resonance (BiaCore), and nanosequencing technologies, has contributed a wealth of new insights into these complex multiprotein mechanisms and clearly accelerated the discovery process. Arguably, the yeast two-hybrid system has been one of the predominant and most powerful tools in this discovery process.
General Methods.- The Two-Hybrid System.- Growth and Maintenance of Yeast.- Media Formulations for Various Two-Hybrid Systems.- Yeast Two-Hybrid Vectors and Strains.- High-Efficiency Transformation of Plasmid DNA into Yeast.- Qualitative and Quantitative Assessment of Interactions.- Strategies for Rescuing Plasmid DNA from Yeast Two-Hybrid Colonies.- False Positives.- Two-Hybrid System and False Positives.- Confirming Yeast Two-Hybrid Protein Interactions Using In Vitro Glutathione-S-Transferase Pulldowns.- Two-Hybrid Interactions Confirmed by Coimmunoprecipitation of Epitope-Tagged Clones.- Applications.- Smad Interactors in Bone Morphogenetic Protein Signaling.- Protein Interactions Important in Eukaryotic Translation Initiation.- Steroid Receptor and Ligand-Dependent Interaction with Coactivator Proteins.- Interaction of Cellular Apoptosis Regulating Proteins with Adenovirus Anti-apoptosis Protein E1B-19K.- Alternative Strategies.- Mammalian Two-Hybrid Assays.- One-Hybrid Systems f hor Detecting Protein-DNA Interactions.- The Split-Hybrid System.- Three-Hybrid Screens.- Three-Hybrid Screens for RNA-Binding Proteins.- Membrane Recruitment Systems for Analysis of Protein-Protein Interactions.
| Erscheint lt. Verlag | 17.5.2001 |
|---|---|
| Reihe/Serie | Methods in Molecular Biology ; 177 |
| Zusatzinfo | XII, 336 p. |
| Verlagsort | Totowa, NJ |
| Sprache | englisch |
| Maße | 152 x 229 mm |
| Themenwelt | Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie |
| Naturwissenschaften ► Biologie ► Mikrobiologie / Immunologie | |
| Naturwissenschaften ► Biologie ► Zellbiologie | |
| ISBN-10 | 0-89603-808-4 / 0896038084 |
| ISBN-13 | 978-0-89603-808-0 / 9780896038080 |
| Zustand | Neuware |
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