Laboratory Methods in Enzymology: Protein Part A

Laboratory Methods in Enzymology: Protein Part A

1st Edition - January 8, 2014
This is the Latest Edition
  • Editor: Jon Lorsch
  • Hardcover ISBN: 9780124200708
  • eBook ISBN: 9780124200975

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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

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

Product details

  • No. of pages: 200
  • Language: English
  • Copyright: © Academic Press 2014
  • Published: January 8, 2014
  • Imprint: Academic Press
  • Hardcover ISBN: 9780124200708
  • eBook ISBN: 9780124200975

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