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

Details

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

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