Methods for analysis of Golgi complex function
Model systems and functional studies -- Imaging-based approaches -- Emerging studies [new frontiers]
Saved in:
| Main Author: | |
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| Other Authors: | |
| Format: | Edited Volume |
| Language: | English |
| Published: |
Burlington
Elsevier Science
2013
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| Series: | Methods in cell biology
118 |
| In: |
Methods in cell biology (118)
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| Volumes / Articles: | Show Volumes / Articles. |
| Subjects: | |
| Online Access: | Verlag, Volltext: http://www.sciencedirect.com/science/book/9780124171640 Verlag, Volltext: http://www.sciencedirect.com/science/bookseries/0091679X/118 
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| Author Notes: | ed. by Franck Perez ... |
Table of Contents:
- Front Cover; Methods for Analysis of Golgi Complex Function; Copyright; Contents; Contributors; Preface; Part 1: Model Systems and Functional Studies; Chapter 1: Analysis of Golgi Complex Functions: In Vitro Reconstitution Systems; Introduction; 1.1. Materials; 1.1.1. COPI coat proteins; 1.1.2. Antibodies; 1.1.3. Reagents; 1.2. Methods; 1.2.1. Preparation of semi-intact cells; 1.2.2. COPI vesicle formation from semi-intact cells; 1.2.3. Ultarstructural analysis of COPI vesicles; 1.3. Discussion and Perspective; Acknowledgments; References
- Chapter 2: RNA Interference Approaches to Examine Golgi Function in Animal Cell CultureOverview of Gene Depletion Methodologies; 2.1. Key Considerations for the Analysis of Organelle Function; 2.2. Strategies for RNAi Experiments; 2.3. Choosing Between siRNA and shRNA; 2.4. Sources of Reagents for RNAi; 2.5. Controls; 2.6. Methods; 2.6.1. Gene depletion using siRNA; 2.6.1.1. Calcium phosphate-mediated delivery; 2.6.1.1.1. Materials; 2.6.1.1.2. Method; 2.6.1.2. Lipid-based transfection; 2.6.1.2.1. Materials; 2.6.1.2.2. Method; 2.6.2. Gene depletion using shRNA; 2.6.2.1. Lentivirus production
- 2.6.2.1.1. Materials2.6.2.1.2. Method; 2.6.2.2. Stable cell line production; 2.6.2.2.1. Materials; 2.6.2.2.2. Method; 2.6.3. Validation of suppression; 2.6.3.1. Immunoblotting; 2.6.3.1.1. Materials; 2.6.3.1.2. Method; 2.6.3.2. Immunofluorescence; 2.6.3.2.1. Materials; 2.6.3.2.2. Method; 2.6.3.3. Quantitative PCR; 2.6.3.3.1. Materials; 2.6.3.3.2. Protocol; 2.6.3.3.2.1. RNA isolation; 2.6.3.3.2.2. Reverse transcription; 2.6.3.3.2.3. Quantitative real-time PCR; 2.6.4. Rescue experiments; 2.7. Discussion; Summary; Acknowledgments; References
- Chapter 3: Trafficking Along the Secretory Pathway in Drosophila Cell Line and Tissues: A Light and Electron Microscopy Ap ...Introduction and Rationale; 3.1. Materials; 3.2. Methods; 3.2.1. Drosophila cultured S2 cells; 3.2.1.1. Protein localization in the early secretory pathway (endogenous or overexpressed); 3.2.1.1.1. Transient transfection protocol; 3.2.1.1.2. Stable transfection protocol; 3.2.1.1.3. Fixation for light microscopy; 3.2.1.1.4. Immunolabeling protocol for S2 cells (Fig.3.1A and B); 3.2.1.1.5. GFP fluorescence (Fig. 3.1A and B); 3.2.1.1.6. Confocal microscopy
- 3.2.1.1.7. EM and IEM (Fig. 3.1C and D)3.2.1.2. RNAi in S2 cells; 3.2.1.3. Transport/secretion assays in S2 cells; 3.2.1.4. Live cell imaging; 3.2.2. Protein localization in Drosophila tissues; 3.2.2.1. Immunofluorescence localization of proteins of the early secretory pathway in whole mount tissues; 3.2.2.1.1. Ovaries (Fig. 3.3C-F); 3.2.2.1.2. Salivary glands (Friggi-Grelin et al., 2006) (Fig. 3.3A); 3.2.2.1.3. Imaginal discs (Dunne, Kondylis, & Rabouille, 2002); 3.2.2.1.4. Visualization of overexpressed proteins; 3.2.2.2. Immunofluorescence on thick sections
- 3.2.2.3. EM and IEM of Drosophila tissues