A guide to protein isolation /

A guide to protein isolation /

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Bibliographic Details
Author / Creator:Dennison, Clive.
Edition:2nd ed.
Imprint:Dordrecht ; Boston : Kluwer Academic Publishers, c2003.
Description:x, 248 p. : ill. ; 24 cm.
Language:English
Series:Focus on structural biology; v. 3
Subject:
Proteins -- Separation.
Proteins -- Separation.
Format: Print Book
URL for this record:http://pi.lib.uchicago.edu/1001/cat/bib/5049829
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ISBN:1402012241 (alk. paper)
Notes:Includes bibliographical references and index.
Table of Contents:
  • Acknowledgements
  • Preface
  • Preface to the 2nd edition
  • Chapter 1. Basic physical concepts applicable to the isolation of proteins
  • Chapter 1 study questions
  • Chapter 2. An overview of protein isolation
  • 2.1. Why do it?
  • 2.2. Properties of proteins that influence the methods used in their study
  • 2.3. The conceptual basis of protein isolation
  • 2.3.1. Where to start?
  • 2.3.2. When to stop?
  • 2.4. The purification table
  • 2.5. Chapter 2 study questions
  • Chapter 3. Assay, extraction and subcellular fractionation
  • 3.1. Buffers
  • 3.1.1. Making a buffer
  • 3.1.2. Buffers of constant ionic strength
  • 3.1.3. Calculating the ionic strength of a buffer
  • 3.2. Assays for activity
  • 3.2.1. Enzyme assays
  • 3.2.1.1. The progress curve
  • 3.2.1.2. The enzyme dilution curve
  • 3.2.1.3. The substrate dilution curve
  • 3.2.1.4. The effect of pH on enzyme activity
  • 3.2.1.5. The effect of temperature on enzyme activity
  • 3.3. Assay for protein content
  • 3.3.1. Absorption of ultraviolet light
  • 3.3.2. The biuret assay
  • 3.3.3. The Lowry assay
  • 3.3.4. The bicinchoninic acid assay
  • 3.3.5. The Bradford assay
  • 3.4. Methods for extraction of proteins
  • 3.4.1. Osmotic shock
  • 3.4.2. Pestle homogenisers
  • 3.4.3. The Waring blendor and Virtis homogeniser
  • 3.4.4. The Polytron/Ultra-Turrax-type homogeniser
  • 3.4.5. Grinding
  • 3.4.6. The Parr bomb
  • 3.4.7. Extrusion under high pressure
  • 3.4.8. Sonication
  • 3.4.9. Enzymic digestion
  • 3.5. Clarification of the extract
  • 3.6. Centrifugal subcellular fractionation
  • 3.6.1. The centrifuge
  • 3.6.2. Principles of centrifugation
  • 3.6.3. Sub-cellular fractionation
  • 3.6.4. Density gradient centrifugation
  • 3.7. Chapter 3 study questions
  • Chapter 4. Concentration of the extract
  • 4.1. Freeze drying
  • 4.1.1. Theoretical and practical considerations in freeze-drying
  • 4.1.2. Some tips on vacuum
  • 4.2. Dialysis
  • 4.2.1. The Donnan membrane effect
  • 4.2.2. Counter-current dialysis
  • 4.2.3. Concentration by dialysis (concentrative dialysis)
  • 4.2.4. Perevaporation
  • 4.3. Ultrafiltration
  • 4.3.1. Desalting or buffer exchange by ultrafiltration
  • 4.3.2. Size fractionation by ultrafiltration
  • 4.4. Concentration/fractionation by salting out
  • 4.4.1. Why ammonium sulfate?
  • 4.4.2. Empirical observations on protein salting out
  • 4.4.3. Three-phase partitioning (TPP)
  • 4.4.3.1. Homogenisation in 30% t-butanol
  • 4.5. Fractional precipitation with polyethylene glycol
  • 4.6. Precipitation with organic solvents
  • 4.7. Dye precipitation
  • 4.8. Chapter 4 study questions
  • Chapter 5. Chromatography
  • 5.1. Principles of chromatography
  • 5.1.1. The effect of particle size
  • 5.1.2. The effect of the mobile phase flow rate
  • 5.1.2.1. Linear and volumetric flow rates
  • 5.2. Equipment required for low pressure liquid chromatography
  • 5.2.1. The column
  • 5.2.2. Moving the mobile phase
  • 5.2.3. Monitoring the effluent and collecting fractions
  • 5.2.4. Refrigeration
  • 5.3. Ion-exchange chromatography (IEC)
  • 5.3.1. Ion-exhcange "resins"
  • 5.3.2. Gradient generators
  • 5.3.3. Choosing the pH
  • 5.3.4. An ion-exchange chromatography run
  • 5.4. Chromatofocusing
  • 5.5. Molecular exclusion chromatography (MEC)
  • 5.5.1. The effect of gel sphere size on V[subscript o]
  • 5.5.2. The manufacture of small, uniform, gel spheres
  • 5.5.3. Determination of MW by MEC
  • 5.5.4. Gels used in MEC
  • 5.5.5. An MEC run
  • 5.6. Hydroxyapatite chromatography
  • 5.6.1. The mechanism of hydroxyapatite chromatography
  • 5.7. Affinity chromatography
  • 5.8. Hydrophobic interaction (HI) chromatography
  • 5.9. HPLC
  • 5.9.1. Concepts and terms relevant to HPLC
  • 5.9.2. Stationary phase materials
  • 5.9.3. Solvent systems
  • 5.9.4. Preparative HPLC
  • 5.10. Chapter 5 study questions
  • Chapter 6. Electrophoresis
  • 6.1. Principles of electrophoresis
  • 6.1.1. The effect of the buffer
  • 6.2. Boundary (Tiselius) electrophoresis
  • 6.3. Paper electrophoresis
  • 6.3.1. Electroendosmosis
  • 6.4. Cellulose acetate membrane electrophoresis (CAM-E)
  • 6.5. Agarose gel electrophoresis
  • 6.6. Starch gel electrophoresis
  • 6.7. Polyacrylamide gel electrophoresis (PAGE)
  • 6.7.1. Disc electrophoresis
  • 6.7.1.1. Isotachophoresis
  • 6.8. SDS-PAGE
  • 6.8.1. An SDS-PAGE zymogram for proteinases
  • 6.9. Pore gradient gel electrophoresis
  • 6.10. Isoelectric focusing
  • 6.10.1. Establishing a pH gradient
  • 6.10.2. Control of buoyancy-driven fluid flow
  • 6.10.3. Applying the sample and measuring the pH gradient
  • 6.10.3.1. An analytical IEF system
  • 6.10.3.2. Preparative IEF
  • 6.11. 2-D Electrophoresis
  • 6.12. Non-linear electrophoresis
  • 6.13. Chapter 6 study questions
  • Chapter 7. Immunological methods
  • 7.1. The structure of antibodies
  • 7.2. Antibody production
  • 7.2.1. Making an antiserum
  • 7.2.2. Monoclonal antibodies
  • 7.3. Immunoprecipitation
  • 7.3.1. Immuno single diffusion
  • 7.3.2. Immuno double diffusion
  • 7.3.2.1. Determination of diffusion coefficients
  • 7.4. Immunoprecipitation methods of historical interest
  • 7.4.1. Mancini radial diffusion
  • 7.4.2. Ouchterlony double diffusion analysis
  • 7.4.3. Immunoelectrophoresis
  • 7.4.3.1. Cross-over electrophoresis
  • 7.4.3.2. Rocket electrophoresis
  • 7.4.3.3. Grabar-Williams immunoelectrophoresis
  • 7.4.3.4. Clarke-Freeman 2-D immunoelectrophoresis
  • 7.5. Amplification methods
  • 7.5.1. Complement fixation
  • 7.5.2. Radioimmunoassay (RIA)
  • 7.5.3. Enzyme amplification
  • 7.5.3.1. Enzyme linked immunosorbent assay (ELISA)
  • 7.5.3.2. Immunoblotting
  • 7.5.4. Immunogold labeling with silver amplification
  • 7.5.5. Colloid agglutination
  • 7.6. Chapter 7 study questions
  • Chapter 8. Some common practical methods
  • 8.1. The Bradford dye-binding assay
  • 8.1.1. Reagents
  • 8.1.2. Procedure
  • 8.3. Methods for concentrating protein solutions
  • 8.3.1. Dialysis against sucrose or PEG
  • 8.3.2. SDS/KCl precipitation
  • 8.3.2.1. Reagents
  • 8.3.2.2. Procedure
  • 8.4. SDS-PAGE
  • 8.4.1. Tris-glycine SDS-PAGE
  • 8.4.1.1. Reagents
  • 8.4.1.2. Procedure
  • 8.4.2. Tris-tricine SDS-PAGE
  • 8.4.2.1. Reagents
  • 8.4.2.2. Procedure
  • 8.5. Serva blue G rapid stain
  • 8.5.1. Reagents
  • 8.5.2. Procedure
  • 8.6. Silver staining of electrophoretic gels
  • 8.6.1. Reagents
  • 8.6.2. Procedure
  • 8.7. Protease zymography
  • 8.7.1. Reagents
  • 8.7.2. Procedure
  • 8.8. Western blotting
  • 8.8.1. Reagents
  • 8.8.2. Procedure
  • 8.9. Fractionation of IgG and IgY
  • 8.9.1. Reagents
  • 8.9.2. Isolation of IgG from rabbit serum
  • 8.9.3. Isolation of IgY from chicken egg yolk
  • 8.10. Enzyme-linked immunosorbent assay (ELISA)
  • 8.10.1. Reagents
  • 8.10.2. Procedure
  • Answers to study questions
  • Chapter 1
  • Chapter 2
  • Chapter 3
  • Chapter 4
  • Chapter 5
  • Chapter 6
  • Chapter 7
  • Further sources of information
  • Index
Gift of the Clinton M. and Dorothy R. Doede Book Fund bookplate
The John Crerar Library bookplate

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