Laboratory Methods in Enzymology: Protein Part A - 1st Edition - ISBN: 9780124200708, 9780124200975

Laboratory Methods in Enzymology: Protein Part A, Volume 536

1st Edition

Serial Volume Editors: Jon Lorsch
eBook ISBN: 9780124200975
Hardcover ISBN: 9780124200708
Imprint: Academic Press
Published Date: 31st January 2014
Page Count: 200
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Table of Contents

Contributors
Miscellaneous
Preface
SECTION I: Protein Protocols
Chapter One: Practical Steady-State Enzyme Kinetics
Abstract

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Measure Initial Rates of the Enzyme-Catalyzed Reaction as a Function of Substrate Concentration

6 Step 2 Determine the Kinetic Parameters (Vmax, kcat, Km)

7 Step 3 Analyze the Mode of Action of an Inhibitor

Chapter Two: Quantification of Protein Concentration Using UV Absorbance and Coomassie Dyes
Abstract

1 Theory

2 Equipment

3 Materials

4 Protocol 1

5 Step 1 Quantification of Protein Using UV Absorbance

6 Protocol 2

7 Step 1 Quantification of Protein Using the Coomassie (Bradford) Assay

Chapter Three: Preparation of Protein Samples for Mass Spectrometry and N-Terminal Sequencing
Abstract

1 Theory

2 Equipment

3 Materials

4 Method A Preparation of Protein Samples for Mass Spectrometry

5 Step A1 Purify the Mitochondria by Metrizamide Gradient Centrifugation and Solubilize Them

6 Step A2 Fractionate the Solubilized Mitochondria by Sucrose Density Gradient Sedimentation

7 Step A3 Separate the Proteins by SDS-PAGE

8 Method B Preparation of Protein Samples for N-Terminal Sequencing

9 Step B1 Prepare Whole Cell Lysates of the Cells

10 Step B2 Affinity Purify the Protein of Interest

11 Step B3 Separate Proteins by SDS-PAGE and Transfer to PVDF Membrane

12 Step B4 Stain the PVDF Membrane and Take It to Your Protein Sequencing Facility

SECTION II: Protein Protocols/Cell Lysis
Chapter Four: Lysis of Mammalian and Sf9 Cells
Abstract

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Resuspend Cells in Lysis Buffer

6 Step 2 Lyse Cells Using a French Press

7 Step 3 Clarify the Cell Lysate

SECTION III: Protein Protocols/Measurement of Protein Synthesis and Decay Rates
Chapter Five: In Vivo [35 S]-Methionine Incorporation
Abstract

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Preparation of Culture

6 Step 2 Lyse Cells and TCA Precipitate Proteins

7 Step 3 Count in Scintillation Counter and Analyze Data

Chapter Six: Pulse-Chase Analysis to Measure Protein Degradation
Abstract

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Methionine Pulse-Chase

6 Step 2 Immunoprecipitation

7 Step 3 Derivation of Protein Half-Life

SECTION IV: Protein Protocols/Methods for Protein Derivitization
Chapter Seven: Labeling of a Protein with Fluorophores Using Maleimide Derivitization
Abstract

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Fluorescent Labeling of Protein by Maleimide Derivitization

6 Step 2 Calculate the Efficiency of Labeling

Chapter Eight: Labeling a Protein with Fluorophores Using NHS Ester Derivitization
Abstract

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Fluorescent Labeling of Protein with 5(6)-FAM, SE Using NHS Ester Derivitization

6 Step 2 Calculate the Efficiency of Labeling

Chapter Nine: Protein Derivitization-Expressed Protein Ligation
Abstract

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Expression of Intein Fusion Proteins

6 Step 2 Cell Harvesting and Lysis

7 Step 3 Binding to Chitin Beads and Linking the Peptide

8 Step 4 Elution and Characterization of Protein

Chapter Ten: Protein Biotinylation
Abstract

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Calculations

6 Step 2 Protein Biotinylation

SECTION V: Protein Protocols/Protein Expression
Chapter Eleven: Small-Scale Expression of Proteins in E. coli
Abstract

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Induction of Heterologous Protein Expression in Small-Scale Bacterial Cultures

6 Step 2 Solubility Analysis of Expressed Heterologous Protein

Acknowledgments

Chapter Twelve: Protein Expression-Yeast
Abstract

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Growth of Yeast Cells

6 Step 2 Lysis of the Yeast Cells

7 Step 3 Purification of the Protein

Chapter Thirteen: Recombinant Protein Expression in Baculovirus-Infected Insect Cells
Abstract

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Perform a Viable Cell Count

6 Step 2 Plaque Purify the Recombinant Baculovirus

7 Step 3 Prepare and Titer a Working Stock of the Recombinant Baculovirus

8 Step 4 Infect Insect Cells with the Recombinant Baculovirus and Produce the Protein of Interest

Chapter Fourteen: Single Cell Cloning of a Stable Mammalian Cell Line
Abstract

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Serial Dilution of Cells

6 Step 2 Grow Single Cells and Analyze Protein Expression

Author Index
Subject Index


Description

The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 530 volumes and 40,000 chapters in the collection, this is an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research, and genetics, just to name a few.

This volume brings together a number of core protocols concentrating on protein, carefully written and edited by experts, including:

  • Pulse-chase analysis to measure protein degradation
  • Labeling a protein with fluorophores using NHS ester derivitization
  • Immunoaffinity purification of proteins
  • Proteolytic affinity tag cleavage
  • Purification of GST-tagged proteins

Key Features

  • Indispensable tool for the researcher
  • Carefully written and edited by experts to contain step-by-step protocols
  • This volume focuses on core protocols involving protein

Readership

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


Details

No. of pages:
200
Language:
English
Copyright:
© Academic Press 2014
Published:
Imprint:
Academic Press
eBook ISBN:
9780124200975
Hardcover ISBN:
9780124200708

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