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

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