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Subnuclear Components
Preparation and Fractionation
- 1st Edition - December 28, 2013
- Editor: G. D. Birnie
- Language: English
- Paperback ISBN:9 7 8 - 1 - 4 8 3 1 - 0 9 7 4 - 9
- eBook ISBN:9 7 8 - 1 - 4 8 3 1 - 0 3 4 0 - 2
Subnuclear Components: Preparation and Fractionation focuses on the isolation of subnuclear components of eukaryotic cells. The book first discusses the isolation of nuclear… Read more
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Request a sales quoteSubnuclear Components: Preparation and Fractionation focuses on the isolation of subnuclear components of eukaryotic cells. The book first discusses the isolation of nuclear envelopes from whole tissues. Topics include nuclear envelope in situ; general consideration and evaluation of isolation methods; and nuclear envelope isolation methods. The text describes the isolation of nucleoli, including the isolation of nucleoli from oocytes, nuclei, and physarum, and isolation of plant nucleoli. The book focuses on the preparation, characterization, and fractionation of chromatin. Emphasis is on the preparation of chromatin from interphase and metaphase cells; contaminants of chromatin; characteristics of isolated chromatin; and dissociation and reassociation of chromatin. The text also discusses fractionation and isolation of histones, including the preparation of individual histone fractions and isolation and purification of the five main histone fractions of calf thymus during one preparation. The book also looks at the preparation and properties of chromatin non-histone proteins, including isolation of nuclear RNA and isolation of DNA from eukaryotic cells. The selection is a good source of data for readers interested in the isolation of subnuclear constituents of eukaryotic cells.
1 Isolation of Animal Cell Nuclei
The Structure and Function of Nuclei
Nuclear Structure
Nuclear Function
Reasons for Isolating Nuclei
Problems Encountered when Isolating Nuclei
Cellular Heterogeneity
Cell Disruption
Isolation Media
Separation of Nuclei from the Brei
Criteria of Purity
Specific Methods for Different Tissues
Lower Eucaryotes
Invertebrates and Amphibia
Tissue-Culture Cells
Vertebrate Organs and Tissues
Tumor Tissues
Conclusions
References
2 Isolation of Nuclear Envelopes from Whole Tissue
The Nuclear Envelope In Situ
Morphology
Cytochemistry
Chemical Composition
General Consideration of Isolation Methods
Tonicity of Isolation Media
Ionic Composition of Isolation Media
pH of Isolation Media
Temperature
Homogenization Techniques
High-Density Sucrose Centrifugation
Isolation of Nuclear Envelopes from Nuclei
Nuclear Envelope Isolation Methods
Evaluation of Isolation Methods
Morphological Assessment
Composition
Enzyme Activities
Biochemical Assessment of Contamination
Conclusions
References
3 Isolation of Nucleoli
Isolation of Nucleoli from Oöcytes
Isolation of Nucleoli from Nuclei
Disrupting Nuclei by Deoxyribonuclease
Disrupting Nuclei by Conication
Disrupting Nuclei by Pressure Methods
Isolation of Nuclei and Nucleoli in Citric Acid
Isolation of Nucleoli by Other Methods
Isolation of Nucleoli from Physarum
Isolation of Plant Nucleoli
Conclusions
References
4 Preparation, Characterization and Fractionation of Chromatin
Preparation of Chromatin from Inter-phase Cells
From Animal Tissues and Cells
From Cells other than Animal Cells
Preparation of Chromatin from Meta-phase Cells
Isolation of Meta-phase Cells
Isolation, Purification and Fractionation of Meta-phase Chromosomes
Contaminants of Chromatin
Contaminants of Cytoplasmic Origin
Contaminants of Nuclear Origin
Characteristics of Isolated Chromatin
Chemical Composition
Physicochemical Properties
Template Activity
Electron Microscopic Studies
Immunological Properties
Fractionation of Chromatin
Features Distinguishing Active and Inactive Chromatin
Fragmentation of Chromatin
Characterization of Chromatin Fractions
Separation of Chromatin Fractions
Conclusions
Dissociation and Reassociation of Chromatin
Partial Deproteinization
Reconstitution
Conclusions
References
5 Fractionation and Isolation of Histones
Isolation and Purification of the Five Main Histone Fractions of Calf Thymus during One Preparation
Details of the Procedure
Comments on the Procedure
Purification of Histone Fractions
Gel Exclusion Chromatography for the Separation of All Five Histones
Preparation of Individual Histone Fractions
Preparation of Histone F1
Preparation of Histone Fractions other than F1
Preparation of the Chicken-Erythrocyte-Specific Histone F2C
Preparation of Whole Unfractionated Histones from Calf Thymus
Preparation of Histones from Tissues Other than Thymus
Characterization of Products
Storage of Histones
References
6 Preparation and Properties of Chromatin Non-Histone Proteins
Biological Relevance of Non-Histone Proteins
Control of Gene Expression
Structure of Chromatin
Post-Synthetic Modifications
Methods for Isolating Non-Histone Proteins
Methods Applied to Dehistoned Chromatins
Methods Applied to Dissociated Chromatins
Selective Extraction Procedures
Fractionation of Non-Histone Proteins
Ion-Exchange Chromatography
Iso-Electric Docusing
Gel Filtration
Characterization of Non-Histone Proteins
Electrophoresis
Amino Acid Composition
Immunospecificity
Factors Affecting the Composition of Non-Histone Proteins
Methodological Artifacts
Contamination by other Cell Constituents
Preparation of Nuclei and Chromatin
Methods Developed in the Author's Laboratory
Materials
Fractionation of Chromatin
One-Dimensional Gel Electrophoresis in Sodium Dodecyl Sulphate
Two-Dimensional Gel Electrophoresis
Fractionation of non-Histone Proteins on a Preparative Scale
Notes on the Methods
Conclusions
References
7 Isolation of Nuclear RNA
Problems Encountered in Isolating Nuclear RNA
Amounts
Contaminants
Degradation
Aggregation
Comparison of Methods
Method for Isolating Nuclear RNA
Details of Procedure
Extraction of Total Cellular RNA
Extraction of Nucleolar RNA
Extraction of Low-Molecular-Weight Nuclear RNA
Assessment of Nuclear RNA Preparations
Amounts Obtained
Contaminants
Degradation and Aggregation
Fractionation of Nuclear RNA
References
8 Isolation of DNA from Eucaryotic Cells
Properties of DNA and Problems Associated with its Isolation
How 'Big' is DNA?
To what Extent is the DNA Duplex Intact
Association of Protein and RNA with DNA
DeoxyriboTiuclease Activity in Ribonuclease and Proteolytic Enzymes
Criteria for the Selection of an Isolation Procedure
Comparison of Procedures Described
Methods of Assessing Size, Integrity and Purity of DNA
Estimation of Molecular Weight
Estimation of Integrity
Estimation of DNA and Contaminants
Isolation of DNA: Procedure I
Description of Procedure I
Analysis of Steps in Procedure I, and Possible Modifications
Properties of Product of Procedure I
Comments on Procedure I
Isolation of DNA: Procedure II
Description of Procedure II
Analysis of Steps in Procedure II
Properties of Product of Procedure II
Isolation of DNA: Procedure III
Description of Procedure III
Analysis of Steps in Procedure III, and Properties of the Product
General Conclusions
References
Index
- No. of pages: 348
- Language: English
- Edition: 1
- Published: December 28, 2013
- Imprint: Butterworth-Heinemann
- Paperback ISBN: 9781483109749
- eBook ISBN: 9781483103402