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Guide to Protein Isolation

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ISBN-10: 1402012241

ISBN-13: 9781402012242

Edition: 2nd 2003 (Revised)

Authors: Clive Dennison

List price: $149.99
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Description:

This text takes the reader on a guided tour through the philosophical and physical foundations of protein isolation. Aimed at a student readership, it should also be very useful to life science researchers faced with the task of isolating a protein for the first time.
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Book details

List price: $149.99
Edition: 2nd
Copyright year: 2003
Publisher: Springer
Publication date: 4/30/2003
Binding: Hardcover
Pages: 249
Size: 6.50" wide x 9.50" long x 0.50" tall
Weight: 1.386
Language: English

Acknowledgements
Preface
Preface to the 2nd edition
Basic physical concepts applicable to the isolation of proteins
Chapter 1 study questions
An overview of protein isolation
Why do it?
Properties of proteins that influence the methods used in their study
The conceptual basis of protein isolation
Where to start?
When to stop?
The purification table
Chapter 2 study questions
Assay, extraction and subcellular fractionation
Buffers
Making a buffer
Buffers of constant ionic strength
Calculating the ionic strength of a buffer
Assays for activity
Enzyme assays
The progress curve
The enzyme dilution curve
The substrate dilution curve
The effect of pH on enzyme activity
The effect of temperature on enzyme activity
Assay for protein content
Absorption of ultraviolet light
The biuret assay
The Lowry assay
The bicinchoninic acid assay
The Bradford assay
Methods for extraction of proteins
Osmotic shock
Pestle homogenisers
The Waring blendor and Virtis homogeniser
The Polytron/Ultra-Turrax-type homogeniser
Grinding
The Parr bomb
Extrusion under high pressure
Sonication
Enzymic digestion
Clarification of the extract
Centrifugal subcellular fractionation
The centrifuge
Principles of centrifugation
Sub-cellular fractionation
Density gradient centrifugation
Chapter 3 study questions
Concentration of the extract
Freeze drying
Theoretical and practical considerations in freeze-drying
Some tips on vacuum
Dialysis
The Donnan membrane effect
Counter-current dialysis
Concentration by dialysis (concentrative dialysis)
Perevaporation
Ultrafiltration
Desalting or buffer exchange by ultrafiltration
Size fractionation by ultrafiltration
Concentration/fractionation by salting out
Why ammonium sulfate?
Empirical observations on protein salting out
Three-phase partitioning (TPP)
Homogenisation in 30% t-butanol
Fractional precipitation with polyethylene glycol
Precipitation with organic solvents
Dye precipitation
Chapter 4 study questions
Chromatography
Principles of chromatography
The effect of particle size
The effect of the mobile phase flow rate
Linear and volumetric flow rates
Equipment required for low pressure liquid chromatography
The column
Moving the mobile phase
Monitoring the effluent and collecting fractions
Refrigeration
Ion-exchange chromatography (IEC)
Ion-exhcange "resins"
Gradient generators
Choosing the pH
An ion-exchange chromatography run
Chromatofocusing
Molecular exclusion chromatography (MEC)
The effect of gel sphere size on V[subscript o]
The manufacture of small, uniform, gel spheres
Determination of MW by MEC
Gels used in MEC
An MEC run
Hydroxyapatite chromatography
The mechanism of hydroxyapatite chromatography
Affinity chromatography
Hydrophobic interaction (HI) chromatography
HPLC
Concepts and terms relevant to HPLC
Stationary phase materials
Solvent systems
Preparative HPLC
Chapter 5 study questions
Electrophoresis
Principles of electrophoresis
The effect of the buffer
Boundary (Tiselius) electrophoresis
Paper electrophoresis
Electroendosmosis
Cellulose acetate membrane electrophoresis (CAM-E)
Agarose gel electrophoresis
Starch gel electrophoresis
Polyacrylamide gel electrophoresis (PAGE)
Disc electrophoresis
Isotachophoresis
SDS-PAGE
An SDS-PAGE zymogram for proteinases
Pore gradient gel electrophoresis
Isoelectric focusing
Establishing a pH gradient
Control of buoyancy-driven fluid flow
Applying the sample and measuring the pH gradient
An analytical IEF system
Preparative IEF
2-D Electrophoresis
Non-linear electrophoresis
Chapter 6 study questions
Immunological methods
The structure of antibodies
Antibody production
Making an antiserum
Monoclonal antibodies
Immunoprecipitation
Immuno single diffusion
Immuno double diffusion
Determination of diffusion coefficients
Immunoprecipitation methods of historical interest
Mancini radial diffusion
Ouchterlony double diffusion analysis
Immunoelectrophoresis
Cross-over electrophoresis
Rocket electrophoresis
Grabar-Williams immunoelectrophoresis
Clarke-Freeman 2-D immunoelectrophoresis
Amplification methods
Complement fixation
Radioimmunoassay (RIA)
Enzyme amplification
Enzyme linked immunosorbent assay (ELISA)
Immunoblotting
Immunogold labeling with silver amplification
Colloid agglutination
Chapter 7 study questions
Some common practical methods
The Bradford dye-binding assay
Reagents
Procedure
Methods for concentrating protein solutions
Dialysis against sucrose or PEG
SDS/KCl precipitation
Reagents
Procedure
SDS-PAGE
Tris-glycine SDS-PAGE
Reagents
Procedure
Tris-tricine SDS-PAGE
Reagents
Procedure
Serva blue G rapid stain
Reagents
Procedure
Silver staining of electrophoretic gels
Reagents
Procedure
Protease zymography
Reagents
Procedure
Western blotting
Reagents
Procedure
Fractionation of IgG and IgY
Reagents
Isolation of IgG from rabbit serum
Isolation of IgY from chicken egg yolk
Enzyme-linked immunosorbent assay (ELISA)
Reagents
Procedure
Answers to study questions
Chapter 1
Chapter 2
Chapter 3
Chapter 4
Chapter 5
Chapter 6
Chapter 7
Further sources of information
Index