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(Ebook PDF) Protein Purification Techniques A Practical Approach 2nd Edition by Simon Roe 0199636737 9780199636730 Full chapter

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Protein Purification Techniques A Practical Approach Second Edition Simon Roe (Editor) Digital Instant Download

Author(s): Simon Roe (Editor)
ISBN(s): 9780585483740, 0585483744
Edition: 2
File Details: PDF, 26.95 MB
Year: 2001
Language: english
SKU: EB-1804108 Category: Tag:

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ISBN 10:         0199636737
ISBN 13: 978-0199636730
Author:          Simon Roe

Proteins are an integral part of molecular and cellular structure and function and are probably the most purified type of biological molecule. In order to elucidate the structure and function of any protein it is first necessary to purify it. Protein purification techniques have evolved over the past ten years with improvements in equipment control, automation, and separation materials, and the introduction of new techniques such as affinity membranes and expanded beds. These developments have reduced the workload involved in protein purification, but there is still a need to consider how unit operations linked together to form a purification strategy, which can be scaled up if necessary. The two Practical Approach books on protein purification have therefore been thoroughly updated and rewritten where necessary. The core of both books is the provision of detailed practical guidelines aimed particularly at laboratory scale purification. Information on scale-up considerations is given where appropriate. The books are not comprehensive but do cover the major laboratory techniques and common sources of protein. Protein Purification Techniques focuses on unit operations and analytical techniques. It starts with an overview of purification strategy and then covers initial extraction and clarification techniques. The rest of the book concentrates on different purification methods with the emphasis being on chromatography. The final chapter considers general scale-up considerations. Protein Purification Applications describes purification strategies from common sources: mammalian cell culture, microbial cell culture, milk, animal tissue, and plant tissue. It also includes chapters on purification of inclusion bodies, fusion proteins, and purification for crystallography. A purification strategy that can produce a highly pure single protein from a crude mixture of proteins, carbohydrates, lipids, and cell debris to is a work of art to be admired. These books (available individually or as a set)are designed to give the laboratory worker the information needed to undertake the challenge of designing such a strategy.
 

Protein Purification Techniques A Practical Approach 2nd Table of contents:

  1. Purification Strategy

    • Key considerations
    • Aims of purification—why?
    • The target protein and contaminants
    • Protein structure
    • Source of the product
    • Key steps in purification
    • Process integration
    • Scale-up
    • Monitoring the purification—assays
    • Time and temperature
    • References

  2. Getting Started

    • Overview of lab equipment
      • Ancillary equipment
      • Detection and analysis equipment
      • Separation equipment
    • Control of pH—buffers—principles, selection, and use
    • Purification strategy
      • Ordering of steps
      • Nature of starting material
      • Storage of material
      • Fermentation products
      • Other sources of proteins
      • Making an extract
      • Separation of particulates
      • Protein concentration
      • Adjusting sample composition between steps
    • Protein lability and structure—implications for purification
      • Introduction
      • Denaturation
      • Catalytic site inactivation
      • Proteolysis
      • Other causes of yield loss
    • References

  3. Analysis of Purity

    • Introduction
    • Considering yield and purity
    • Scope of the methods included
    • Total protein quantitation
      • Ultraviolet absorption protein assay
      • Lowry (Folin-Ciocalteau) protein assay
      • Bradford (Coomassie Brilliant Blue) dye-binding protein assay
      • Bicinchoninic acid protein assay
      • Colloidal gold protein assay
      • General comments
    • Specific quantitation
      • Activity assays
      • Binding assays
    • Detection of impurities
      • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
      • Detection of protein bands in electrophoresis gels
      • Analysis by isoelectric focusing (IEF)
      • Glycoprotein analysis
      • Western blotting
      • Chromatographic methods
    • Other biochemical and biophysical methods
      • Protein mass spectrometry
    • References

  4. Clarification Techniques

    • Introduction
    • Sedimentation
      • Principles of sedimentation
      • Gravity sedimentation
    • Centrifugal sedimentation
      • Centrifuge equipment
      • Laboratory centrifuge test work
      • Suspension conditioning
    • Filtration
      • Background
      • Principles of dead-end filtration
      • Laboratory scale filtration equipment
      • Application of filtration
    • Membrane processes
      • Background
      • Principles of operation
      • Membrane equipment
      • Membrane selection
      • Membrane operation
    • Choice of clarification process

  5. Cell Disintegration and Extraction Techniques

    • Overview
      • Stability of the released protein
      • Location of target protein within the cell
      • Yield and kinetics of the process
      • Continuous or batch disruption
      • The need to consider subsequent steps
      • Assessing the extent of disruption
      • Marker substances for cell disruption
      • Containment of the process
      • Scale-up
    • Methods of disruption
      • Pre-treatment of material
      • General procedure notes
      • Mixers and blenders
      • Coarse grinding: pestle and mortar
      • Fine grinding: the bead mill
      • Homogenization
      • Ultrasonication
      • Heat shock
      • Freezing and thawing
      • Osmotic shock
      • Lytic enzymes
      • Chemical treatments
      • Detergents
      • Solvents
    • Conclusions: choice of methods
    • References

  6. Concentration of the Extract

    • Introduction
    • Addition of a dry matrix polymer
    • Ultrafiltration
      • Equipment
      • Operation
      • Other applications of ultrafiltration
    • Freeze-drying or lyophilization
    • Removal of salts and exchange of buffer
      • Dialysis
      • Diafiltration
      • Gel filtration
    • Purification and concentration by precipitation
      • Precipitation by alteration of the pH
      • Precipitation by decreasing the ionic strength
      • Precipitation by increasing the ionic strength (salting-out)
      • Precipitation by organic solvents
      • Precipitation by organic polymers
      • Precipitation by denaturation
    • Aqueous two-phase partitioning
      • Equipment and materials
      • Optimization
      • Using phase partitioning in a purification procedure
      • Affinity partitioning
    • Acknowledgements
    • References

  7. Separation on the Basis of Chemistry

    • Introduction
    • Chemical interaction between surfaces
    • Basic properties of proteins
    • Properties of the solvent
    • Properties of chromatography media
      • Ion exchange chromatography
      • Hydrophobic interaction chromatography
      • Reversed-phase chromatography
      • Charge-transfer chromatography
      • Covalent chromatography
    • Design, optimization, and scale-up of a chromatographic purification procedure
    • Acknowledgements
    • References

  8. Chromatography on the Basis of Size

    • Introduction
      • Principle
      • Applications of size exclusion chromatography
    • Materials
      • Equipment
      • Mobile phase
      • Non-ideal size exclusion
    • Methods
      • Flow rates
      • Preparation and packing
      • Equilibrium
      • Sample preparation and application
      • Evaluation and calibration
      • Separation of proteins
      • Column cleaning and storage
      • Troubleshooting
    • References

  9. Purification by Exploitation of Activity

    • Introduction
    • Design and preparation of affinity adsorbents
      • Choice of ligand
      • Selection of the matrix
      • Choice of spacer
      • Activation and coupling chemistry
      • Estimation of ligand concentration
    • Use of affinity adsorbents
      • Initial questions
      • Selection of conditions for operation
      • Estimation of capacity
      • Ligand efficiency
      • Application to protein purification
    • Immunopurification
      • Antibody selection
      • Immobilization of antibodies on CNBr-activated Sepharose
      • Procedures for immunopurification
    • References

  10. Scale-Up Considerations

  • Scale-up of purification processes and unit operations
    • Choice of scale
    • Overview of critical issues
    • Practical aspects of scale-up

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