Cloning, Gene Expression, and Protein Purification: Experimental Procedures and Process Rationale / Edition 1

by Charles Hardin, Jennifer Edwards, Andrew Riell, David Presutti, William Miller

Overview

On the forefront of modern scientific innovation, Cloning, Gene Expression and Protein Purification: Experimental Procedures and Process Rationale effectively doubles as a laboratory manual for students and a reference book for professional researchers. Designed for advanced undergraduate and beginning graduate students in molecular biology, this unique combination lecture/laboratory resource presents detailed protocols for the multi-step process involved in isolating a gene, cloning and characterizing it, expressing its encoded protein, and purifying and characterizing the protein's basic physical properties. This manageable volume includes both theoretical background and practical procedures and is structured around twenty experiments that demonstrate how to prepare, manipulate, and analyze plasmids, produce fusion proteins in bacteria, and purify these proteins based on unique chemical properties or substrate affinities. The book describes advanced topics such as the use of antibodies and the techniques developed to transform their structures, as well as combinatorial approaches designed to manipulate the structure and functions of proteins and nucleic acids. Supplemental literature provides a variety of theoretical explanations encouraging a more intuitive understanding of the experimental mechanisms and behaviors of the chemical participants, while also giving students the tools needed to become "capable proactive researchers." Features:
· Emphasizes electrophoresis, Southern and Western blotting, and combinatorial techniques
· Defines clear reaction mechanisms; stipulates the functions of reagents; and helps students think about the precise consequences of solution and procedural manipulations
· Discusses fluorophores, and solvent effects on protein structure
· Characterizes plasmids, cDNAs, and antibody probes (available from ATCC) in research literature
· Includes carefully selected primary source research literature and articles from current vendor literature
· Contains a glossary of unfamiliar phrases and jargon; important summary statements and conclusions are italicized
· Provides an alphabetized list of common reagents for rapid reference
· Offers an extensive index of concepts and terms
· Categorizes helpful and distinctive information into five types of supplemental literature: Innovation/ Insight, Theory/Principle, Process Rationale, Vendor Literature, and Alternative Approaches

Product Details

ISBN-13:	9780195132946
Publisher:	Oxford University Press
Publication date:	03/01/2001
Edition description:	New Edition
Pages:	448
Product dimensions:	8.54(w) x 10.96(h) x 0.85(d)

About the Author

Charles Hardin is an Associate Professor of Biochemistry at North Carolina State University. Jennifer Pinczes recently completed her Master's of Science in biochemistry at North Carolina State University. Andrew Riell is a computer specialist and consultant with NetAspects, Inc. William Miller is William Neals Reynolds Professor of Biochemistry at North Carolina State University. David Presutti is an Adjunct Visiting Professor of Biochemistry at North Carolina State University. Dominique Robertson is an Associate Professor of Botany at North Carolina State University.

Table of Contents

Introductory UnitIntroductory Lecture - Introduction to the Biochemical LaboratoryTheory: Course DescriptionTheory: "Central Dogma of Molecular Biology"Theory: Laboratory SafetyTheory: The Scientific Method: Surviving Recipe MentalityTheory: Proactive TroubleshootingTheory: Introduction to the Biotechnology LaboratoryTheory: Error Analysis and Assay SensitivityTheory: Treatment of Analytical DataTheory: Concentration and Temperature Effects on pKaIntroductory Lab 1 - Basic Biochemical Techniques I: Pipet Calibration and Solution PreparationProcess: PipetsIntroductory Lab 2 - Basic Techniques II: Absorbance Spectroscopy and Protein Concentration DeterminationsProcess: AMP and Tryptophan Absorbance Spectra; Sample CalculationsTheory: Absorption Data for the Nucleoside MonophosphatesProcess: Absorption Spectra Data for the Aromatic Amino Acids at pH 6; UV Absorption Characteristics of the Aromatic Amino Acids. Selected Extinction CoefficientsProcess: BCA Assay Sample DataInnov.: Measurement of Protein in 20 SecondsPart I - Nucleic Acids & CloningUnit 1Lecture 1 - DNA IsolationTheory: Subcloning ProcedureInnov.: The pET Bacterial Plasmid System (Novagen)Lab 1.1 - Media Preparation; Bacterial Growths; Plasmid Minipreps; HindIII Digestion of DNA, Commercial Bacteriophage h DNA BstEII Digest Size StandardsProcess: pUR278 and p2D Restriction MapsProcess: Cloning the myo-3 Gene from C. elegans and Construction of an Expression VectorProcess: C. elegans myo-3 Gene in pUR288Vend. Lit.: Restriction Enzymes HindIII and BstEII; h DNA DigestsProcess: Phage h BstEII DigestLab 1.2 - Agarose Gel ElectrophoresisExercises: Restriction MappingUnit 2Lecuture 2 - Construction of Recombinant PlasmidsInnov.: Protecting and Manipulating Large DNA SubstratesInnov.: Yeast of Burden—Yoking the YACLab 2.1 - Extraction and Cleanup of DNA Bands Cut from Agrose Gels, Quantitation of Yields and Ligation of myo-3 HindIII DNA Insert Fragment into Linearized B-gal Plasmid DNAVend. Lit.: Gibco BRL (TM) T4 DNA LigaseVend. Lit.: DNA Purification Kit (NaI/Glass Bead Method)Alt. App.: The Use of B-Agarase to Recover DNA from Gel SlicesAlt. App.: GELase TMUnit 3Lecture 3 - The Polymerase Chain ReactionInnov.: Polymerase Chain Reaction Used for Antigen Detection; Immuno-PCR: Very Sensitive Antigen Detection by Means of Specific Antibody-DNA ConjugatesLab 3.1 - Polymerase Chain Reaction Test for myo-3 Gene Insert OrientationUnit 4Lecture 4 - Transcription of Genomic DNA & Analysis of the Resulting mRNAsAlt. App.: Isolation of Total RNA from E. coli CellsAlt. App.: Promega TM PolyATractTM System 1000Alt. App.: Electrophoresis and Northern Blotting of RNAUnit 5Lecture 5 - Transformation and Gene ExpressionInnov.: How Cells Respond to StressLab 5.1 - Preparation of Fresh Transformation - Competent CellsAlt. App.: Ultracomp TM Transformation KitLab 5.2 - Colony Immunoblotting to Screen for TransformantsAlt. App.: The QIAexpressionist, QIAGENTMUnit 6Lecture 6 - Analysis of DNA or RNA by Duplex Hybridization: DNA Isolation, Labeling, and ProbingInnov.: Reduction of Background Problems in Nonradioactive Northern and Southern Blot Analyses Enables Higher Sensitivity than 32P-Based HybridizationLab 6.1 - Labeling of DNA and Probe Construction from Cloned C. elegans myo-3 Gene; Quantitation of DNA ConcentrationVend. Lit.: Digoxigenin Labeling of DNA: Genius TM Nucleic Acid Labeling SystemLab 6.2 - Isolation of C. elegans Genomic DNA, Quantitation of DNA Concentration, and Digestion to Extract the myo-3 GeneLab 6.3 - Southern BlottingPart 2 - Protein PurificationUnit 7Lecture 7 - Protein PurificationTheory: Preparation and Handling of Biological Macromolecules for CrystallizationTheory: Solution STructure of Biomacromolecules in Ionic SolutionsTheory: Solubility as a Function of Protein Structure and Solvent ComponentsTheory: Dominant Forces in Protein FoldingAlt. App.: Hydrophobic Interaction ChromatographyAlt. App.: Centriprep Microconcentrators for Small Volume Concentrations; Centricon-3 and 100Lab 7.1 - The Protein Purifier: A Learning Aid from PharmaciaLab 7.2 - Induction and Purification of B-Galactosidase Fusion Protein from BacteriaLab 7.3 - Gel Filtration of Molecular Weight Standards and Protein FractionationProcess: Gel Filtration and ChromatographyVend. Lit.: Sephadex and SephacrylVend. Lit.: Sigma TM Gel Filtration Molecular Weight MarkersLab 7.4 - Mciroplate B-Galactosidase Assay to Determine Fractions Containing Fusion Protein; MW DeterminationProcess: Time Course Assay of B-GalactosidaseVend. Lit.: B-Galactosidase SubstratesInnov.: Luminescent Reporter Gene Assays for Luciferase and B-Galactosidase Using a Liquid Scintillation CounterLab 7.5 - Ion Exchange Column ChromatographyProcess: Ion Exchange ChromatographyTheory: The Isoelectric Point: Protein Charge Neutrality at a Particular pHAlt. App.: Ion-Pair ChromatographyAlt. App.: HPLC: Ion Exchange and Reverse Phase Methods; Literature SourcesLab 7.6 - Affinity Chromatography and Microplate B-Galactosidase Assays to Determine Fractions Containing Fusion ProteinProcess: Affinity ChromatographyProcess: Affinity Chromatography: One Step Purification of Hybrid Proteins Carrying Fused B-Galactosidase ActivityLab 7.7 - BCA Protein Concentration Assays and B-Galactosidase Assays to Construct an Enzyme Purification TableUnit 8Lecture 8 - Discontinuous Gel Electrophoresis, Protein Mobilities and Apparent Size DeterminationProcess: Discontinuous Gel Electrophoresis & Protein Size DeterminationLab 8.1 - Discontinuous SDS Gel ElectrophoresisUnit 9Lecture 9 - Immunochemical TechniquesInnov.: Immunochemical TechniquesInnov.: The Enzyme Linked Immunosorbent Assay (ELISA)Innov.: How the Immune System Learns About SelfInnov.: Making Monoclonal Antibodies That Won't Fight BackLab 9.1 - Western BlottingProcess: ImmunoblottingProcess: Western Blots Using Stained Protein GelsUnit 10Lecture 10 - Combinatorial Biochemical TechnologyInnov.: Examples of Combinatorial TechniquesInnov.: Making Antibody Fragments Using Phage Display LibrariesInnov.: Building a Better EnzymeInnov.: The ImmunoZAP Cloning and Expression SystemAppendicesPart 1 Terms ListPart 2 Terms ListLaboratory ReagentsAbbreviations ListCopyright AcknowledgementsSuggested ScheduleSupplies RequiredIndex(Abbreviations:Innov., Innovation/InsightTheory, Theory/PrinciplesProcess, Process RationaleVend. Lit., Vendor LiteratureAlt. App., Alternative Approach