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About this book
About this book
Investigators who have identified and cloned a gene of interest often want to isolate and characterize the protein product, yet the methods required are notoriously difficult for the inexperienced. This manual has been adapted from a course held at the Cold Spring Harbour Laboratory in New York, 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 manual 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. The book should be of use to specialists in genetics, microbiology, neuroscience, and cell biology, who wish to develop expertise in working with proteins.
Part 1 Purification of calmodulin. Experiments: activity assays - assay of calmodulin fractions; preparation of a tissue extract; bulk fractionation; ion-exchange chromatography; hydrophobic interaction chromatography; characterization of calmodulin - calculation of recovery; characterization of calmodulin - electrophoresis; proteolytic digestion; reverse-phase HPLC; physical analysis of calmodulin; chemical analysis of calmodulin. Preparation of reagents. Part 2 Purification of transcription factor AP-1 from HeLa cells. Experiments: preparation of a nuclear extract from HeLa cells; gel filtration chromatography with Sephacryl S-300 HR; sequence-specific DNA affinity chromatography; DNase 1 footprinting; gel-mobility-shift assay; preparation of heparin-sepharose CL-2B; preparation of reagents. Part 3 Purification of recombinant protein overproduced in escherichia coli. Experiments: breakage of E. coli cells and preparation of inclusion bodies; solubilization, refolding, and ion-exchange chromatography of the inclusion body pellet (o32); polyethyleneimine precipitation and immunoaffinity chromatography of the soluble extract (core RNA polymerase o32 complex); quantitation and summary of preparation; protein characterization. Protocol development trials - purification of o32 from a bacterial overexpresser; preparation of reagents. Part 4 Solubilization and purification of the rat liver insulin receptor. Experiments: isolation of plasma membranes from rat liver; solubilization of insulin receptor from membranes; lectin affinity chromatography of solubilized receptors; insulin affinity chromatography of partially purified receptors; cross-linking of insulin receptors with [125I] insulin; insulin-stimulated insulin receptor autophosphorylation; analysis of insulin receptor glycosylation. Preparation of reagents.