Laboratory Methods in Enzymology: Protein Part A

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