Laboratory Methods in Enzymology: Protein Part C

1st Edition, Volume 541 - April 10, 2014
Latest edition
Editor: Jon Lorsch
Language: English
Hardback ISBN:
9 7 8 - 0 - 1 2 - 4 2 0 1 1 9 - 4
eBook ISBN:
9 7 8 - 0 - 1 2 - 4 2 0 1 7 8 - 1

In this volume we have brought together a number of core protocols concentrating on Protein, carefully written and edited by ex… Read more

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

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

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Laboratory Methods in Enzymology: Protein Part C

Fuel your confidence!

Jon Lorsch

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