Laboratory Methods in Enzymology: Protein Part C
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Laboratory Methods in Enzymology: Protein Part C

1st Edition - March 22, 2014

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  • Editor: Jon Lorsch
  • eBook ISBN: 9780124201781
  • Hardcover ISBN: 9780124201194

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Description

In this volume we have brought together a number of core protocols concentrating on Protein, carefully written and edited by experts.

Key Features

  • Indispensable tool for the researcher
  • Carefully written and edited by experts to contain step-by-step protocols
  • In this volume we have brought together a number of core protocols concentrating on Protein

Readership

Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists

Table of Contents

    • Miscellaneous
    • Preface
    • Section I: Protein Protocols/Protein Precipitation
    • Chapter One. TCA Precipitation
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1A Trichloroacetic Acid Precipitation
      • 6 Step 1B Deoxycholate-Trichloroacetic Acid Precipitation
      • References
    • Section II: Protein Protocols/Protein Pull-Down Methods
    • Chapter Two. Coimmunoprecipitation of Proteins from Yeast
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1 Preparation of Whole Cell Lysates
      • 6 Step 2 Normalization of Cell Lysates
      • 7 Step 3 Coimmunoprecipitation
      • 8 Step 4 Wash and Elute the Immunoprecipitates
      • 9 Step 5 Analysis of Immunoprecipitations
      • References
      • Related Literature
      • Referenced Protocols in Methods Navigator
    • Chapter Three. Coupling Antibody to Cyanogen Bromide-Activated Sepharose
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1 Preparation of Antibody and Resin
      • 6 Step 2 Coupling the Antibody to the Resin
      • 7 Step 3 Quench the Reaction
      • 8 Step 4 Wash the Resin
      • References
    • Chapter Four. Analysis of Protein–Protein Interactions by Coimmunoprecipitation
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1 Isolation of the Protein of Interest by Immunoprecipitation
      • 6 Step 2 Detection of the Binding Partner by Immunoblotting
      • References
      • Referenced Protocols in Methods Navigator
    • Section III: Protein Protocols/Protein Purification
    • Chapter Five. Use and Application of Hydrophobic Interaction Chromatography for Protein Purification
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1 Column Equilibration
      • 6 Step 2 Column Loading
      • 7 Step 3 Product Elution
      • 8 Step 4 Adsorbent Regeneration and Sanitization
      • References
      • Source References
    • Chapter Six. Hydroxyapatite Chromatography: Purification Strategies for Recombinant Proteins
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1 Purification Protocol Screening by Linear Salt Gradient
      • 6 Step 2 Purification Protocol using a Step Gradient
      • 7 Step 3 Purification Protocol using a Step Gradient and Simplified Analytics
      • 8 Step 4 Offline pH Measurement and Calcium ion Analysis
      • 9 Step 5 SEC Profile for the Collected mAb Fraction and Regeneration Fraction
      • References
    • Chapter Seven. Salting out of Proteins Using Ammonium Sulfate Precipitation
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1 Removal of Proteins Marginally Soluble in (NH4)2SO4
      • 6 Step 2 Precipitation of the Protein of Interest
      • References
      • Referenced Protocols in Methods Navigator
    • Chapter Eight. Using Ion Exchange Chromatography to Purify a Recombinantly Expressed Protein
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1 Equilibration of the Column
      • 6 Step 2 Binding of the Protein Sample
      • 7 Step 3 Removal of Unbound Proteins
      • 8 Step 4 Elution of the Bound Protein
      • References
      • Referenced Protocols in Methods Navigator
    • Chapter Nine. Gel Filtration Chromatography (Size Exclusion Chromatography) of Proteins
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1 Standardization of the Gel Filtration Column
      • 6 Step 2 Determination of the Sizes of Protein Species in a Sample
      • References
      • Referenced Protocols in Methods Navigator
    • Section IV: Protein Protocols/Purification of Membrane Proteins
    • Chapter Ten. Expression and Purification of Membrane Proteins
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1 Transformation of E. coli
      • 6 Step 2 Cultivation of E. coli – Screening for the Optimal Expression Conditions
      • 7 Step 3 Scale-Up Expression of a Membrane Protein Using the Optimal Expression Conditions
      • 8 Step 4 Screening Detergents to Determine Optimal Solubilization of Membrane Protein
      • 9 Step 5 Scale-up the Solubilization of the Membrane Protein
      • 10 Step 6 Purification of Membrane Proteins Using Ni-NTA Superflow
      • References
      • Related Literature
      • Referenced Protocols in Methods Navigator
    • Chapter Eleven. Explanatory Chapter: Choosing the Right Detergent
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Using Detergents with Polyacrylamide Gel Electrophoresis
      • 6 Using Detergents in Chromatograpy
      • 7 Using Detergents with Optical Spectroscopy Techniques
      • 8 Using Detergents with Mass Spectrometry Techniques
      • 9 Using Detergents with Nuclear Magnetic Resonance (NMR)
      • 10 Using Detergents in Protein Crystallization
      • References
    • Section V: Protein Protocols/SDS PAGE
    • Chapter Twelve. One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE)
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1 Casting an SDS-PAGE Gel: Resolving Gel
      • 6 Step 2 Casting an SDS-PAGE Gel: Stacking Gel
      • 7 Step 3 Running an SDS-PAGE Gel
      • References
      • Referenced Protocols in Methods Navigator
    • Chapter Thirteen. Coomassie Blue Staining
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1 Stain a Gel Using Coomassie Blue
      • 6 Step 2 Destain the Gel to Reduce Background Staining
      • Source References
    • Chapter Fourteen. Silver Staining of SDS-polyacrylamide Gel
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1 Fix the Gel
      • 6 Step 2 Stain the Gel
      • 7 Step 3 Preserve the Gel
      • References
    • Section VI: Protein Protocols/Standard in vitro Assays for Protein-Nucleic Acid Interactions
    • Chapter Fifteen. Standard In Vitro Assays for Protein–Nucleic Acid Interactions – Gel Shift Assays for RNA and DNA Binding
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1 Radiolabeling the Nucleic Acid Probe
      • 6 Step 2 Bind Protein and Nucleic Acid
      • 7 Step 3 Preparation of Polyacrylamide Gel
      • 8 Step 4 Loading and Running Gel
      • 9 Step 5 Analysis of Gel
      • References
      • Source References
    • Chapter Sixteen. Protein Filter Binding
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1 Assemble Binding Reactions
      • 6 Step 2 Quantify Binding
      • 7 Step 3 Process Binding Data
      • References
      • Source References
    • Section VII: Protein Protocols/Troubleshooting Protein Expression
    • Chapter Seventeen. Explanatory Chapter: Troubleshooting Recombinant Protein Expression: General
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1 Monitoring E. coli cell Growth Before Induction
      • 6 Step 2 Induction of Expression
      • 7 Step 3 Monitoring E. coli Cell Growth After Induction
      • 8 Step 4 Measurement of Protein Production
      • 9 Step 5 Further Troubleshooting in E. coli
      • 10 Step 6 Eukaryotic Expression Systems
      • References
      • Source References
    • Chapter Eighteen. Explanatory Chapter: Troubleshooting Protein Expression: What to do When the Protein is not Soluble
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1 Centrifuge the Culture
      • 6 Step 2 Lyse the Cells
      • 7 Step 3 Remove the Cell Debris
      • 8 Step 4 Analyze Protein Expression by SDS-PAGE
      • 9 Step 5 Troubleshooting the Lack of Soluble Protein Expressed
      • References
      • Referenced Protocols in Methods Navigator
    • Section VIII: Protein Protocols/Western Blotting
    • Chapter Nineteen. Western Blotting using Chemiluminescent Substrates
      • Abstract
      • 1 Theory
      • 2 Equipment
      • 3 Materials
      • 4 Protocol
      • 5 Step 1: Protein Transfer to a Membrane
      • 6 Step 2: Western Blot Detection using a Chemiluminescent Substrate
      • References
      • Source References
    • Author Index
    • Subject Index

Product details

  • No. of pages: 296
  • Language: English
  • Copyright: © Academic Press 2014
  • Published: March 22, 2014
  • Imprint: Academic Press
  • eBook ISBN: 9780124201781
  • Hardcover ISBN: 9780124201194

About the Serial Volume Editor

Jon Lorsch

Affiliations and Expertise

Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA

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