Cold Spring Harbor Laboratory Press

Strategies for Protein Purification and Characterization A Laboratory Course Manual

By Daniel R. Marshak, Cold Spring Harbor Laboratory; James T. Kadonaga, Department of Biol- ogy, University of California, San Diego; Richard R. Burgess, University of Wisconsin, Madison, and Mark W. Knuth, Promega Corporation, Madison, Wisconsin; William A. Brennan, Jr., Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, and Sue-Hwa Lin, University of Texas M.D. Anderson Cancer Center

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.

C O N T E N T S Foreword by James E. Rothman Introduction How to Use This Manual UNIT I: PURIFICATION OF CALMODULIN Introduction Experiment l: 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 Experiment 8: Proteolytic Digestion Experiment 9: Reverse-phase HPLC Experiment 10: Physical Analysis of Calmodulin Experiment 11: ChemicalAnalysis of Calmodulin Preparation of Reagents References UNIT II: PURIFICATION OF TRANSCRIPTION FACTOR AP-1 FROM HELA CELLS Introduction Experiment l: Preparation of a Nuclear Extract from HeLa Cells Experiment 2: Gel Filtration Chromatography with Sephacryl S-300 HR Experiment 3: Sequence-specific DNA Affinity Chromatography Experiment 4: 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 Introduction Experiment 1: Breakage of E. coli Cells and Preparation of Inclusion Bodies Experiment 2: Solubilization, Refolding, and Ion-exchange Chroma- tography of the Inclusion Body Pellet (t~ 32) Experiment 3: Polyethyleneimine Precipitation and Immunoaffin~ Chromatography of the Soluble Extract (Core RNA Polymerase-o J- Complex) Experiment 4: Quantitation and Summary of Preparation Experiment 5: Protein Characterization Protocol Development Trials: Purification of ~32 from a Bacterial Overexpresser Preparation of Reagents References UNIT IV: SOLUBILIZATION AND PURIFICATION OF THE RAT LIVER INSULIN RECEPTOR Introduction Experiment 1: Isolation of Plasma Membranes from Rat Liver Experiment 2: Solubilization of Insulin Receptor from Membranes Experiment 3: Lectin Affinity Chromatography of Solubilized Re- ceptors Experiment 4: Insulin Affinity Chromatography of Partially Purified Receptors Experiment 5: Cross-linking of Insulin Receptors with [125I] Insulin Experiment 6: Insulin-stimulated Insulin Receptor Autophosphoryla- tion Experiment 7: Analysis of Insulin Receptor Glycosylation Preparation of Reagents References Appendices

1996, 396 pp., illus., appendices, indexes Plastic comb binding $85 ISBN 0-87969-385-1

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Copyright �9 1996 by Cold Spring Harbor Laboratory Press

Volume 3 N u m b e r 1

July/August 1996 Pages 1-60

Review

A Biochemist's View of Long-term Potentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Erik D. Roberson, Joey D. English, and J. David Sweatt

Research papers

c-Fos Induction in the Rat Nucleus of the Solitary Tract Correlates with the Retention and Forgetting of a Conditioned Taste Aversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Thomas A. Houpt, Jennifer M. Philopena, Tong H. Joh, and Gerard P. Smith

Transient Expression of c-Fos in Rat Amygdala During Training Is Required for Encoding Conditioned Taste Aversion Memory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Raphael Lamprecht and Yadin Dudai

Examination of the Role of cGMP in Long-term Potentiation in the CA I Region of the Hippocampus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 David K. Selig, Michele R. Segal, Dezhi Liao, Robert C. Malenka, Roberto Malinow, Roger A. Nicoll, and John E. Lisman

Conditioned Visual Flight Orientation in Drosophila: Dependence on Age, Practice, and Diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Aike Guo, Liu Li, Xia Shou-zhen, Feng Chun-hua, Reinhard Wolf, and Martin Heisenberg

Cover c-Fos translation in the amygdala during or immediately after CTA training is essential for encoding taste aversion memory. Shown are sites of microinjection in this study reported by Lamprecht and Dudai (see pp. 31-41). (Brain sections are adapted from the atlas of Paxinos and Watson 1986.)