Intermediate filament associated proteins /

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...

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Detalles Bibliográficos
Clasificación:Libro Electrónico
Otros Autores: Wilson, Katherine L. (Editor ), Sonnenberg, Arnoud (Editor )
Formato: Electrónico eBook
Idioma:Inglés
Publicado: Waltham, MA : Academic Press, an imprint of Elsevier, 2016.
Edición:First edition.
Colección:Methods in enzymology ; v. 569.
Temas:
Proteins.
Intermediate filament proteins.
Intermediate Filament Proteins
Proteins
Prot�eines.
Prot�eines des filaments interm�ediaires.
protein.
SCIENCE > Life Sciences > Biochemistry.
Intermediate filament proteins
Internet Resources.
Acceso en línea:Texto completo
Texto completo
Tabla de Contenidos:
  • Front Cover
  • Intermediate Filament Associated Proteins
  • Copyright
  • Contents
  • Contributors
  • Preface
  • Part I: Identification and Biochemical Analysis of Lamin-Associated Proteins
  • Chapter One: BioID Identification of Lamin-Associated Proteins
  • 1. Introduction
  • 1.1. Nuclear Lamina
  • 1.2. Protein-Protein Interactions
  • 1.3. BioID
  • 2. BioID Method
  • 2.1. Construction of a BioID-Fusion Protein
  • 2.2. Cell Choice
  • 2.3. Functional Assessment of the Fusion Protein
  • 2.4. Establishing a Stable BioID Cell Line
  • 2.4.1. Materials.
  • 2.4.2. Generation and Validation of BioID-Fusion Protein
  • 2.4.3. Generate Cells That Stably Express the BioID-Fusion Protein
  • 2.5. Large-Scale BioID Pull-Down to Identify Interacting Proteins
  • 2.5.1. Materials
  • 2.5.2. Cell Lysis
  • 2.5.3. Affinity Purification of Biotinylated Proteins
  • 2.6. Reagents and Solutions
  • 3. Anticipated Results
  • 4. Conclusions
  • Acknowledgment
  • References
  • Chapter Two: Proximity-Dependent Biotin Identification (BioID) in Dictyostelium Amoebae
  • 1. Introduction
  • 1.1. Lamins and Their Partners: The Challenge
  • 1.2. Advantages and Limitations of BioID.
  • 1.2.1. Limitations of BioID
  • 1.3. Adaptation of BioID to the Dictyostelium System
  • 1.4. Overview of BioID Protocol in Dictyostelium
  • 2. Materials
  • 2.1. Materials for Strain Generation
  • 2.2. Materials for Cell Growth and Isolation of Nuclei
  • 2.2.1. Cell Preparation
  • 2.2.2. Cell Lysis and Isolation of Nuclei
  • 2.2.3. Solubilization of Nuclear Material and Isolation of Biotinylated Proteins
  • 2.3. Materials for Light Microscopy
  • 3. Methods
  • 3.1. Generate Strains Expressing the BirA-R118G-Fused Protein of Interest
  • 3.1.1. Cell Growth and Electrotransformation.
  • 3.1.2. Clonal Selection with Blasticidin S
  • 3.1.3. Clonal Selection with G418
  • 3.2. Isolate Nuclei, Prepare Lysates, and Purify Biotinylated Proteins
  • 3.2.1. Cell Preparation
  • 3.2.2. Cell Lysis and Isolation of Nuclei
  • 3.2.3. Solubilization of Nuclei and Isolation of Biotinylated Proteins
  • 3.3. Light Microscopy
  • 3.3.1. Fixation and Fluorescence Microscopy of Whole Cells
  • 3.3.2. Fixation and Fluorescence Microscopy of Isolated Nuclei
  • 3.4. Electrophoresis and Immunoblotting
  • 4. Conclusions
  • References.
  • Chapter Three: Purification and Structural Analysis of LEM-Domain Proteins
  • 1. Introduction
  • 2. ��Divide and Conquer�� Approach to Study LEM-Domain Proteins
  • 2.1. The LEM Domain Common to LAP2, Emerin, and MAN1
  • 2.2. The C-Terminal Domain Specific to LAP2�
  • 2.3. The WH and UHM Domains in MAN1
  • 3. Analysis of Predicted Unstructured Regions in LEM-Domain Proteins
  • 3.1. Production and Purification of Predicted Unstructured Regions of Emerin and MAN1
  • 3.1.1. Emerin Nucleoplasmic Region
  • 3.1.2. The N-Terminal Nucleoplasmic Region of MAN1.

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