Read more
This volume discusses various basic and advanced methods and protocols that have been proven to be successful among certain bacterial species, or a family of species, in type III secretion systems (T3S system). The chapters in this book cover topics such as: site-directed mutagenesis and its application in studying the interactions of T3S components; use of transcriptional control to increase expression and secretion of heterologous proteins in T3S systems; fractionation techniques to examine effector translocation; detecting immune responses to T3S systems; mouse immunization with purified needle proteins from T3S systems and the characterization of the immune responses to these proteins; and detection of protein interactions in T3S systems using yeast 2-hybrid analysis. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Practical and comprehensive, Type 3 Secretion Systems: Methods and Protocols is a valuable resource for anyone interested in learning about the fascinating and ever-changing T3S systems.
List of contents
Introduction to Type III Secretion Systems.- Site-Directed Mutagenesis and its Application in Studying the Interactions of T3S Components.- Blue Native Protein Electrophoresis to Study the T3S System using Yersinia pestis as a Model.- In Vivo Photo-Cross-Linking to Study T3S Interactions Demonstrated using the Yersinia pestis T3S System.- Isolation of Type III Secretion System Needle Complexes by Shearing.- Use of Transcriptional Control to Increase Expression and Secretion of Heterologous Proteins in T3S Systems.- Characterization of Type Three Secretion System Translocator Interactions with Phospholipid Membranes.- Analysis of Type III Secretion System Secreted Proteins.- Fractionation Techniques to Examine Effector Translocation.- Measurement of Effector Protein Translocation using Phosphorylatable Epitope Tags and Phospho-Specific Antibodies.- A TAL-Based Reporter Assay for Monitoring Type III-Dependent Protein Translocationin Xanthomonas.- Subcellular Localization of Pseudomonas syringae pv. tomato Effector Proteins in Plants.- A Method for Characterizing the Type III Secretion System's Contribution to Pathogenesis: Homologous Recombination to Generate Yersinia pestis Type III Secretion System Mutants.- Detecting Immune Responses to Type III Secretion Systems.- Recombinant Expression and Purification of the Shigella Translocator IpaB.- Expression and Purification of N-Terminally His-Tagged Recombinant Type III Secretion Proteins.- Mouse Immunization with Purified Needle Proteins from Type III Secretion Systems and the Characterization of the Immune Response to these Proteins.- Identification of the Targets of Type III Secretion System Inhibitors.- Detection of Protein Interactions in T3S Systems using Yeast 2-Hybrid Analysis.
Summary
This volume discusses various basic and advanced methods and protocols that have been proven to be successful among certain bacterial species, or a family of species, in type III secretion systems (T3S system). The chapters in this book cover topics such as: site-directed mutagenesis and its application in studying the interactions of T3S components; use of transcriptional control to increase expression and secretion of heterologous proteins in T3S systems; fractionation techniques to examine effector translocation; detecting immune responses to T3S systems; mouse immunization with purified needle proteins from T3S systems and the characterization of the immune responses to these proteins; and detection of protein interactions in T3S systems using yeast 2-hybrid analysis. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Practical and comprehensive, Type 3 Secretion Systems: Methods and Protocols is a valuable resource for anyone interested in learning about the fascinating and ever-changing T3S systems.
