Contents
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