© 1996 • 396 pp., illus., appendices, index
This book has been produced using print on demand technology.
Spiral binding • $48.00 • ISBN  978-087969385-5

Investigators who have identified and cloned a gene of interest often want to isolate and characterize the protein product, yet the methods required are no-toriously tricky for the inexperienced. For the past four years, a course has been held at Cold Spring Harbor Laboratory to teach scientists how to execute the major protein techniques by applying them to four distinct, representative types of molecule: a regulatory protein, a DNA-binding protein, a recombinant protein, and a membrane-bound receptor. This course has now been adapted in the form of a laboratory manual that covers a variety of bulk fractionation, electrophoretic, and chromatographic techniques. Step-by-step protocols are accompanied by troubleshooting advice and guidance on generalizing the techniques for other classes and types of protein. The emphasis throughout is on strategies for purification and characterization rather than automated instrumental analysis.

After years of rigorous testing, these techniques are robust and reliable, and are presented here with the clarity and completeness for which Cold Spring Harbor manuals are celebrated. The book is invaluable for specialists in genetics, microbiology, neuroscience, and cell biology who wish to develop expertise in working with proteins.

  "The book is a laboratory-course manual targeted at scientists who have no or very limited knowledge about protein purification and has evolved from years of teaching the corresponding course at Cold Spring Harbor Laboratory…. In my opinion, the authors have covered an excellent set of projects and methods, enabling a beginner to get a feel for the basic principles in protein biochemistry…. Nevertheless, this book is a superb manual for a course in principles in protein biochemistry for advanced graduate students or interested faculty members either at a university/research institute or for those attending a course such as that mentioned above at Cold Spring Harbor Laboratory."
      —Trends in Cell Biology

Foreword

Introduction

Protein Properties That Can Be Used as Handles for Purification; Types of Molecular Interactions and Variables That Affect Them; Types of Separation Methods; References

How To Use This Manual

UNIT I: PURIFICATION OF CALMODULIN (D.R. Marshak)

Introduction

Experiment 1: Activity Assays: Assay of Calmodulin Fractions

Experiment 2: Preparation of a Tissue Extract

Experiment 3: Bulk Fractionation

Experiment 4: Ion-exchange Chromatography

Experiment 5: Hydrophobic Interaction Chromatography

Experiment 6: Characterization of Calmodulin: Calculation of Recovery

Experiment 7: Characterization of Calmodulin: Electrophoresis

Polyacrylamide Gel Electrophoresis in Sodium Dodecyl Sulfate; Native Polyacrylamide Gel Electrophoresis without Detergent; Urea Gradient Polyacrylamide Gel Electrophoresis and the Analysis of Folding

Experiment 8: Proteolytic Digestion

Digestion of Calmodulin with Cyanogen Bromide; Digestion of Calmodulin with Trypsin

Experiment 9: Reverse-phase HPLC

Experiment 10: Physical Analysis of Calmodulin

Experiment 11: Chemical Analysis of Calmodulin

Preparation of Reagents

References

UNIT II: PURIFICATION OF TRANSCRIPTION FACTOR AP-1 FROM HELA CELLS (J.T. Kadonaga)

Introduction

Experiment 1: Preparation of a Nuclear Extract from HeLa Cells

Experiment 2: Gel Filtration Chromatography with Sephacryl S-300 HR

How to Pour a Gel Filtration Resin; Sephacryl S-300 HR Chromatography of the HeLa Nuclear Extract

Experiment 3: Sequence-specific DNA Affinity Chromatography

Purification of Oligonucleotides by Preparative Gel Electrophoresis; Preparation of the DNA Affinity Resin; Proper Use of Nonspecific Competitor DNAs in Affinity Chromatography; Procedure for Affinity Chromatography

Experiment 4: DNase I Footprinting

Preparation of a DNase I Footprinting Probe; DNase I Footprinting

Experiment 5: Gel Mobility-shift Assay

Experiment 6: Preparation of Heparin-Sepharose CL-2B

Preparation of Reagents

References

UNIT III: PURIFICATION OF A RECOMBINANT PROTEIN OVERPRODUCED IN ESCHERICHIA COLI (R.R. Burgess and M.W. Knuth)

Introduction

Experiment 1: Breakage of E. coli Cells and Preparation of Inclusion Bodies

Experiment 2: Solubilization, Refolding, and Ion-exchange Chromatography of the Inclusion Body Pellet (s32)

Experiment 3: Polyethyleneimine Precipitation and Immunoaffinity Chromatography of the Soluble Extract (Core RNA Polymerase-s32 Complex)

Fractionation of the Soluble Extract by PEI Precipitation; Immunoaffinity Chromatography

Experiment 4: Quantitation and Summary of Preparation

Protein Determination; Quantitative SDS Gel Staining and Scanning; Quantitative Western Dot Blots; Enzyme Assay; Balance Sheet; Protein Purification Summary Table and Summary Photograph of Main Fractions

Experiment 5: Protein Characterization

UV Spectrum of the Pure ProteinÄA280nm/A260nm; Determination of the Extinction Coefficient (Method of Scopes); Estimation of Purity by Scanning an Overloaded Stained SDS Gel; Assessment of Homogeneity by Mobility on Nondenaturing Gel Electrophoresis

Protocol Development Trials: Purification of s32 from a Bacterial Overexpresser

Rapid Dot Blot Western Assays; Is the s32 Soluble or Insoluble; How Much Sarkosyl Is Needed to Dissolve the Inclusion Body Pellet; How Much Polyethyleneimine Is Needed to Precipitate s32 and RNA Polymerase; How Much Salt Is Needed to Elute s32 and RNA Polymerase from the PEI Pellet; How Long Does It Take to Remove the Sarkosyl by Dialysis; What Dialysis Conditions Will Result in the Highest Yields of Refolded, Soluble Monomeric s32; How Long Does It Take for Protein to Bind to the Immunoaffinity Resin; Alternative Procedures for Refolding Denatured s32; Strategies for Rapid Development of a New Protocol for Purification from Bacterial Overexpresser (Assumes No Disulfide Bridges)

Preparation of Reagents

References

UNIT IV: SOLUBILIZATION AND PURIFICATION OF THE RAT LIVER INSULIN RECEPTOR (W.A. Brennan, Jr. and S.-H. Lin)

Introduction

Experiment 1: Isolation of Plasma Membranes from Rat Liver

Neville's Procedure for the Isolation of Liver Plasma Membranes; Alternative Procedure for the Isolation of a Crude Membrane Fraction from Tissues; Determination of Protein Concentration in the Presence of Interfering Substances; Determination of the Number of Insulin Receptors in a Membrane; Determination of the Affinity of Insulin Receptor for Insulin

Experiment 2: Solubilization of Insulin Receptor from Membranes

Solubilization of the Insulin Receptor and Assessment of Activity; Screening Detergents for Membrane Protein Solubilization

Experiment 3: Lectin Affinity Chromatography of Solubilized Receptors

Experiment 4: Insulin Affinity Chromatography of Partially Purified Receptors

Insulin Affinity Chromatography; Coupling of Ligands to Activated Beads

Experiment 5: Cross-linking of Insulin Receptors with [125I] Insulin

Experiment 6: Insulin-stimulated Insulin Receptor Autophosphorylation

Experiment 7: Analysis of Insulin Receptor Glycosylation

Preparation of Reagents

References

Appendices

1. Measurement of pH; 2. Preparation of Buffers; 3. Measurement of Conductivity;
4. Fractionation with Solid Ammonium Sulfate; 5. Polyacrylamide Gel Electrophoresis of Proteins in Sodium Dodecyl Sulfate: Electrophoresis, Staining of Gels, Sample Preparation by Precipitation; 6. Oxidation of Proteins with Performic Acid; 7. Isolation of Phosphopeptides Using Iminodiacetic Acid (Iron-chelating) Sepharose 6B; 8. Isolation and Separation of Peptide Fragments for Internal Sequence Analysis of Proteins; 9. Recommended Reading