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DNA sensors and inflammasomes /
| Clasificación: | Libro Electrónico |
|---|---|
| Otros Autores: | |
| Formato: | Electrónico eBook |
| Idioma: | Inglés |
| Publicado: |
Cambridge, MA :
Academic Press,
2019.
|
| Colección: | Methods in enzymology ;
v. 625. |
| Temas: | |
| Acceso en línea: | Texto completo |
Tabla de Contenidos:
- Front Cover; DNA Sensors and Inflammasomes; Copyright; Contents; Contributors; Preface; Chapter One: Intercellular communication in the innate immune system through the cGAS-STING pathway; 1. Introduction; 2. cGAS-STING signaling; 2.1. Inter-cellular communication; 2.2. Type 1 IFN; 3. Immuno-signaling to neighboring cells through gap junctions; 3.1. cGAMP in virus particles; 3.2. Extracellular vesicles; 4. Exosomes and immune responses to virus infections; 5. Exosomes and immune responses to bacterial infections; 6. Concluding remarks; References
- Chapter Two: Analysis of human cGAS activity and structure1. Introduction; 2. A genetic assay for screening cGAS activity using bacteria; 2.1. Assay setup and considerations; 2.2. Protocol; 2.3. Future applications; 3. Crystallization and structural determination of the human cGAS-DNA complex; 3.1. Preparation of human cGAS protein and DNA; 3.1.1. Equipment; 3.1.2. Reagents; 3.1.3. Protocol; 3.2. Assays to assess human cGAS-DNA complex formation; 3.2.1. Equipment; 3.2.2. Reagents; 3.2.3. Protocol; 3.3. Human cGAS-DNA crystallization protocol; 3.3.1. Equipment; 3.3.2. Reagents; 3.3.3. Protocol
- 3.4. Human cGAS-DNA structure determination3.4.1. Equipment; 3.4.2. Reagents; 3.4.3. Protocol; 4. Crystallization of a human cGAS-DNA-ligand complex; 4.1. Crystal soaking and human cGAS-DNA-ligand crystallization; 4.2. Protocol for ligand soaking and crystal harvest; 4.2.1. Reagents; 4.2.2. Protocol; 5. Summary; Acknowledgments; References; Chapter Three: Chemical synthesis, purification, and characterization of 3-5-linked canonical cyclic dinucleotides (CDNs); 1. Introduction; 2. Synthesis of crude CDNs; 2.1. Chemicals; 2.2. Equipment; 2.3. Synthesis procedure for crude c-AMP-CMP
- 3. Purification of crude CDNs3.1. Preparation of HPLC buffers; 3.2. Purification protocol for crude c-AMP-CMP; 3.3. HPLC purification of crude c-AMP-CMP; 4. Characterization of CDNs; 4.1. Materials and equipment; 4.2. MS analysis of c-AMP-CMP fractions; 4.3. NMR analysis of c-AMP-CMP; 5. Summary and prospects; Acknowledgments; References; Chapter Four: An SPR-based analysis of cGAS substrate KD and steady-state KM values; 1. Introduction; 2. Consideration for the immobilization of proteins, multimers, and the cGASds-DNA complex; 3. Considerations for SPR-based enzymology; 4. Protocols
- 4.1. Protein reagents4.2. Chemical reagents; 4.3. Equipment; 4.4. Chip preparation and protein capture; 4.5. cGAS-ligand binding studies; 4.5.1. Preparation of samples; 4.5.2. Details of injections and data collection; 4.5.3. Data analysis; 4.6. cGAS activity assays; 4.6.1. Preparation of samples; 4.6.2. Details of injections and data collection; 4.6.3. Data analysis; 5. Summary; References; Chapter Five: A pyrophosphatase-coupled assay to monitor the NTase activity of cGAS; 1. Introduction; 2. Materials; 2.1. Chemicals, materials and enzymes; 2.2. Buffers and assay reagents