Rab GTPases Methods and Protocols 2nd Edition by Guangpu Li, Nava Segev ISBN B0B6548YS3 9781071613467

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ISBN 10: B0B6548YS3
ISBN 13: 9781071613467 
Author: Guangpu Li, Nava Segev

This second edition volume expands on the previous edition with a discussion of new research and discoveries in the Rab field. Chapters in this book cover topics such as new information on Rab regulation and localization; interaction; function; and diseases. 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. 

Cutting-edge and comprehensive, Rab GTPases: Methods and Protocols, Second Edition is a valuable resource for scientists working in the fields of Rab and other small GTPases, and beyond

Rab GTPases Methods and Protocols 2nd Edition Table of contents:

Chapter 1: Newer Methods Drive Recent Insights into Rab GTPase Biology — An Overview

1. Introduction

2. Rab Regulation and Localization (Chapters 2–8)

3. Rab Interactions (Chapters 9–12)

4. Rab Function (Chapters 13–18)

5. Rab Dysfunction and Disease (Chapters 19–21)

6. *Future Perspectives: How New Technologies Can Benefit the Rab Field?
   References

Chapter 2: Rab29 Fast Exchange Mutants — Characterization of a Challenging Rab GTPase

1. Introduction

2. Materials
    2.1 Purifying Rab29 and Its Mutants
    2.2 MANT-GDP Assay

3. Methods
    3.1 Purification of Rab29 Proteins
    3.2 MANT-GDP Assay

4. Notes
   References

Chapter 3: High-Throughput Assay for Profiling the Substrate Specificity of Rab GTPase-Activating Proteins

1. Introduction

2. Materials
    2.1 Reagents and Chemicals
    2.2 Supplies
    2.3 Proteins
    2.4 Buffers
    2.5 Chromatographic Columns
    2.6 Instruments
    2.7 Software

3. Methods
    3.1 GTP Loading and Preparation of 2x Solutions
    3.2 GAP Assay and Measurement
    3.3 Kinetic Data Analysis
     3.3.1 Initial Velocity Approach
     3.3.2 Exponential Fit Approach
     3.3.3 Integrated Michaelis–Menten Approach

4. Notes
   References

Chapter 4: Detecting Endogenous Rab8 Activation

1. Introduction

2. Materials
    2.1 Generation of GST-Fusion Probes
    2.2 Pull-Down of Active Rab8 from Cell Lysates
    2.3 SDS-PAGE and Immunoblotting

3. Methods
    3.1 Molecular Cloning of GST-OCRL-RBD and GST-PI3K-RasBD
    3.2 Bacterial Expression of GST-OCRL-RBD and GST-PI3K-RasBD
    3.3 Rab8 Activation Assay and Immunoblotting

4. Notes
   References

Chapter 5: Testing the Phenotypic Effects of a Rab Chimera that Resolves Exchange Factor Specificity from Effector Specificity

1. Introduction

2. Materials
    2.1 Bgl2 Secretion Assay
    2.2 Electron Microscopy Study
    2.3 GFP-Snc1 Microscopy
    2.4 Effector Localization

3. Methods
    3.1 Bgl2 Assay (Fig. 1)
    3.2 Electron Microscopy (Fig. 2)
    3.3 Fluorescence Microscopy and Quantitative Localization of GFP-Snc1 (Fig. 3)
    3.4 Effector Localization (Fig. 4)

4. Notes
   References

Chapter 6: Profiling Structural Alterations During Rab5 Nucleotide Exchange by HDX-MS

1. Introduction

2. Materials
    2.1 Protein Expression — Bacteria
    2.2 Protein Expression — Insect Cell
    2.3 Protein Purification
    2.4 HDX-MS

3. Methods
    3.1 Protein Expression of Rab5
    3.2 Purification of Rab5
    3.3 Protein Expression of Rabex5:Rabaptin5
    3.4 Protein Purification Rabex5:Rabaptin5
    3.5 HDX-MS

4. Notes
   References

Chapter 7: CLEM Characterization of Rab8 and Associated Membrane Trafficking Regulators at Primary Cilium Structures

1. Introduction

2. Materials
    2.1 Cells and Culture Media
    2.2 Chemicals, Buffers, and Solutions
    2.3 Equipment
    2.4 Fluorescence and Electron Microscopies

3. Methods
    3.1 Cell Culture and Stable Cell Line Generation
    3.2 Light Microscopy
    3.3 TEM Sample Preparation
    3.4 Identifying the Target Cell in the Resin Block
    3.5 Ultra-Thin Sectioning Using an Ultramicrotome
    3.6 Electron Microscopy Imaging
    3.7 Alignment and Processing of Fluorescence and EM Images

4. Notes
   References

Chapter 8: Imaging of Spatial Cycling of Rab GTPase in the Cell

1. Introduction

2. Major Outcomes and Possible Uses

3. Materials
    3.1 Construction of Plasmids
    3.2 Mammalian Cell Culture and Transfection Reagents
    3.3 Generation of PRA1 and TRAPPC4 Knockdown Stable Cell Lines
    3.4 In Cellulo Prenylation Experiment
    3.5 Live Cell Imaging

4. Methods
    4.1 Design and Cloning of the Target Construct
    4.2 Generation of PRA1 and TRAPPC4 Knockdown Cell Lines
    4.3 Microscopy and Imaging
     4.3.1 FRAP Experiments
     4.3.2 FLAP Experiments
    4.4 In Cellulo Prenylation Assay

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Tags: Guangpu Li, Nava Segev, Rab GTPases Methods, Protocols