Laboratory Methods in Enzymology: Protein Part C - 1st Edition - ISBN: 9780124201194, 9780124201781

Laboratory Methods in Enzymology: Protein Part C, Volume 541

1st Edition

Serial Volume Editors: Jon Lorsch
eBook ISBN: 9780124201781
Hardcover ISBN: 9780124201194
Imprint: Academic Press
Published Date: 10th April 2014
Page Count: 296
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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

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


Details

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

Ratings and Reviews


About the Serial Volume Editors

Jon Lorsch Serial Volume Editor

Affiliations and Expertise

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