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Intermediate Filament Associated Proteins
1st Edition - January 14, 2016
Editors: Katherine L. Wilson, Arnoud Sonnenberg
Hardback ISBN:9780128034699
9 7 8 - 0 - 1 2 - 8 0 3 4 6 9 - 9
eBook ISBN:9780128034903
9 7 8 - 0 - 1 2 - 8 0 3 4 9 0 - 3
Intermediate Filament Associated Proteins, the latest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by… Read more
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Intermediate Filament Associated Proteins, the latest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in intermediate filament associated proteins and contains sections on such topics as lamin-associated proteins, intermediate filament-associated proteins and plakin, and other cytoskeletal cross-linkers.
Continues the legacy of this premier serial with quality chapters authored by leaders in the field
Covers research methods in intermediate filament associated proteins and contains sections on such topics as lamin-associated proteins, intermediate filament-associated proteins and plakin, and other cytoskeletal cross-linkers
Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists.
Preface
Part I: Identification and Biochemical Analysis of Lamin-Associated Proteins
Chapter One: BioID Identification of Lamin-Associated Proteins
Abstract
1 Introduction
2 BioID Method
3 Anticipated Results
4 Conclusions
Acknowledgment
Chapter Two: Proximity-Dependent Biotin Identification (BioID) in Dictyostelium Amoebae
Abstract
1 Introduction
2 Materials
3 Methods
4 Conclusions
Chapter Three: Purification and Structural Analysis of LEM-Domain Proteins
Abstract
1 Introduction
2 “Divide and Conquer” Approach to Study LEM-Domain Proteins
3 Analysis of Predicted Unstructured Regions in LEM-Domain Proteins
4 LEM-Domain Proteins with Their Partners
5 Concluding Remarks
Acknowledgments
Chapter Four: Purification and Structural Analysis of SUN and KASH Domain Proteins
Abstract
1 Introduction
2 Purification of SUN Proteins and SUN–KASH Complexes
3 Structural Analysis of Human SUN2 and SUN2–KASH1/2 Complexes
4 Conclusions and Pitfalls
Acknowledgments
Chapter Five: Purification of Lamins and Soluble Fragments of NETs
Abstract
1 Introduction
2 Purification Parameters
3 Lamin A Purification
4 NET Fragment Purification
5 Gel Extraction of Proteins
6 Conclusions and Potential Applications
Acknowledgments
Chapter Six: Simple Separation of Functionally Distinct Populations of Lamin-Binding Proteins
Abstract
1 Introduction
2 Cell Culture (with or without Transfection) and Harvesting
3 Separate Nuclei from Cytoplasm
4 Fractionate Nuclei to Separate “Easy-” Versus “Sonication-Dependent” Proteins
5 Analysis by Immunoprecipitation and Immunoblotting
6 Conclusions
Acknowledgments
Part II: General Methods for Analysis of Intermediate Filament-Associated Proteins
Chapter Seven: Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins
Abstract
1 Introduction
2 Recombinant Proteins
3 Fluorescence Measurement of Soluble EGFP
4 Expression of EGFP-Fusion Proteins in Mammalian Cells
5 Analysis of the Interaction of EGFP-Fusion Recombinant Plakins with IF Proteins
6 Concluding Remarks
Acknowledgments
Chapter Eight: Functional Analysis of Keratin-Associated Proteins in Intestinal Epithelia: Heat-Shock Protein Chaperoning and Kinase Rescue
Abstract
1 Introduction
2 Isolation of Keratin Intermediate Filaments for Functional Assays
3 Detection of Chaperoning Activity on Keratin Intermediate Filaments, Relative to Cytosolic Activity
4 Refolding of aPKC by Hsp70/40 in Keratin Intermediate Filaments
5 Supplementation Assays Using Cytosol (S) and Isolated Keratin Intermediate Filaments (kP)
6 Summary
Acknowledgments
Chapter Nine: Purification of Protein Chaperones and Their Functional Assays with Intermediate Filaments
Abstract
1 Introduction
2 Purification of sHSPs
3 Purification of IF Proteins
4 Analysis of sHSP and IF Proteins Interaction
Acknowledgments
Chapter Ten: Purification and Structural Analysis of Plectin and BPAG1e
Abstract
1 Introduction
2 Purification of Fragments of Plectin and BPAG1e for Structural Analysis
3 Structural Analysis of Plectin and BPAG1e
4 Analysis of Multi-Domain Structures Combining MX, DEER, and SAXS
5 Conclusion
Acknowledgments
Chapter Eleven: Purification and Structural Analysis of Desmoplakin
Abstract
1 Introduction
2 Purification and Structural Analysis of DPNT
3 Purification and Structural Analysis of DPCT
4 Future Directions
Acknowledgments
Chapter Twelve: Degradation of the Intermediate Filament Family by Gigaxonin
Abstract
1 Introduction
2 Gigaxonin Expression and Localization
3 Analyzing Gigaxonin Role in IF Organization
4 Future Perspectives
Acknowledgments
Part III: Functional and Genetic Analysis of the Plakin and Other Cytoskeletal Cross-Linkers
Chapter Thirteen: Functional and Genetic Analysis of Plectin in Skin and Muscle
Abstract
1 Introduction
2 Main Challenges
3 Tools
4 Methods, Pearls, and Pitfalls
5 Conclusions
Acknowledgments
Chapter Fourteen: Functional and Genetic Analysis of Epiplakin in Epithelial Cells
Abstract
1 Introduction
2 Main Considerations
3 Methods: Pearls and Pitfalls
4 Conclusions
Acknowledgments
Chapter Fifteen: In Vitro Model of the Epidermis: Connecting Protein Function to 3D Structure
Abstract
1 Introduction
2 Historical Perspective
3 Cellular Constituents of 3D Raft Cultures
4 3D Raft Cultures
5 Experimental Manipulation of the Organotypic Raft Cultures
6 Analysis of Organotypic Raft Cultures and Other Methods
7 Conclusions
Acknowledgments
Chapter Sixteen: Functional Analysis of Periplakin and Envoplakin, Cytoskeletal Linkers, and Cornified Envelope Precursor Proteins
Abstract
1 Introduction
2 Implications of Plakin Domain Structure to Functional Analysis
3 Analysis of the Plakin Expression and Localization in Cell Culture Models
4 Ablation of Periplakin Expression by RNA Interference
5 Identification of Periplakin Interaction Partners