Pub. Date:
Oxford University Press
Cloning, Gene Expression, and Protein Purification: Experimental Procedures and Process Rationale / Edition 1

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


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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: 10.60(w) x 8.20(h) x 1.00(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 Unit
Introductory Lecture - Introduction to the Biochemical Laboratory
Theory: Course Description
Theory: "Central Dogma of Molecular Biology"
Theory: Laboratory Safety
Theory: The Scientific Method: Surviving Recipe Mentality
Theory: Proactive Troubleshooting
Theory: Introduction to the Biotechnology Laboratory
Theory: Error Analysis and Assay Sensitivity
Theory: Treatment of Analytical Data
Theory: Concentration and Temperature Effects on pKa
Introductory Lab 1 - Basic Biochemical Techniques I: Pipet Calibration and Solution Preparation
Process: Pipets
Introductory Lab 2 - Basic Techniques II: Absorbance Spectroscopy and Protein Concentration Determinations
Process: AMP and Tryptophan Absorbance Spectra; Sample Calculations
Theory: Absorption Data for the Nucleoside Monophosphates
Process: Absorption Spectra Data for the Aromatic Amino Acids at pH 6; UV Absorption Characteristics of the Aromatic Amino Acids. Selected Extinction Coefficients
Process: BCA Assay Sample Data
Innov.: Measurement of Protein in 20 Seconds
Part I - Nucleic Acids & Cloning
Unit 1
Lecture 1 - DNA Isolation
Theory: Subcloning Procedure
Innov.: 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 Standards
Process: pUR278 and p2D Restriction Maps
Process: Cloning the myo-3 Gene from C. elegans and Construction of an Expression Vector
Process: C. elegans myo-3 Gene in pUR288
Vend. Lit.: Restriction Enzymes HindIII and BstEII; h DNA Digests
Process: Phage h BstEII Digest
Lab 1.2 - Agarose Gel Electrophoresis
Exercises: Restriction Mapping
Unit 2
Lecuture 2 - Construction of Recombinant Plasmids
Innov.: Protecting and Manipulating Large DNA Substrates
Innov.: Yeast of Burden—Yoking the YAC
Lab 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 DNA
Vend. Lit.: Gibco BRL (TM) T4 DNA Ligase
Vend. Lit.: DNA Purification Kit (NaI/Glass Bead Method)
Alt. App.: The Use of B-Agarase to Recover DNA from Gel Slices
Alt. App.: GELase TM
Unit 3
Lecture 3 - The Polymerase Chain Reaction
Innov.: Polymerase Chain Reaction Used for Antigen Detection; Immuno-PCR: Very Sensitive Antigen Detection by Means of Specific Antibody-DNA Conjugates
Lab 3.1 - Polymerase Chain Reaction Test for myo-3 Gene Insert Orientation
Unit 4
Lecture 4 - Transcription of Genomic DNA & Analysis of the Resulting mRNAs
Alt. App.: Isolation of Total RNA from E. coli Cells
Alt. App.: Promega TM PolyATractTM System 1000
Alt. App.: Electrophoresis and Northern Blotting of RNA
Unit 5
Lecture 5 - Transformation and Gene Expression
Innov.: How Cells Respond to Stress
Lab 5.1 - Preparation of Fresh Transformation - Competent Cells
Alt. App.: Ultracomp TM Transformation Kit
Lab 5.2 - Colony Immunoblotting to Screen for Transformants
Alt. App.: The QIAexpressionist, QIAGENTM
Unit 6
Lecture 6 - Analysis of DNA or RNA by Duplex Hybridization: DNA Isolation, Labeling, and Probing
Innov.: Reduction of Background Problems in Nonradioactive Northern and Southern Blot Analyses Enables Higher Sensitivity than 32P-Based Hybridization
Lab 6.1 - Labeling of DNA and Probe Construction from Cloned C. elegans myo-3 Gene; Quantitation of DNA Concentration
Vend. Lit.: Digoxigenin Labeling of DNA: Genius TM Nucleic Acid Labeling System
Lab 6.2 - Isolation of C. elegans Genomic DNA, Quantitation of DNA Concentration, and Digestion to Extract the myo-3 Gene
Lab 6.3 - Southern Blotting
Part 2 - Protein Purification
Unit 7
Lecture 7 - Protein Purification
Theory: Preparation and Handling of Biological Macromolecules for Crystallization
Theory: Solution STructure of Biomacromolecules in Ionic Solutions
Theory: Solubility as a Function of Protein Structure and Solvent Components
Theory: Dominant Forces in Protein Folding
Alt. App.: Hydrophobic Interaction Chromatography
Alt. App.: Centriprep Microconcentrators for Small Volume Concentrations; Centricon-3 and 100
Lab 7.1 - The Protein Purifier: A Learning Aid from Pharmacia
Lab 7.2 - Induction and Purification of B-Galactosidase Fusion Protein from Bacteria
Lab 7.3 - Gel Filtration of Molecular Weight Standards and Protein Fractionation
Process: Gel Filtration and Chromatography
Vend. Lit.: Sephadex and Sephacryl
Vend. Lit.: Sigma TM Gel Filtration Molecular Weight Markers
Lab 7.4 - Mciroplate B-Galactosidase Assay to Determine Fractions Containing Fusion Protein; MW Determination
Process: Time Course Assay of B-Galactosidase
Vend. Lit.: B-Galactosidase Substrates
Innov.: Luminescent Reporter Gene Assays for Luciferase and B-Galactosidase Using a Liquid Scintillation Counter
Lab 7.5 - Ion Exchange Column Chromatography
Process: Ion Exchange Chromatography
Theory: The Isoelectric Point: Protein Charge Neutrality at a Particular pH
Alt. App.: Ion-Pair Chromatography
Alt. App.: HPLC: Ion Exchange and Reverse Phase Methods; Literature Sources
Lab 7.6 - Affinity Chromatography and Microplate B-Galactosidase Assays to Determine Fractions Containing Fusion Protein
Process: Affinity Chromatography
Process: Affinity Chromatography: One Step Purification of Hybrid Proteins Carrying Fused B-Galactosidase Activity
Lab 7.7 - BCA Protein Concentration Assays and B-Galactosidase Assays to Construct an Enzyme Purification Table
Unit 8
Lecture 8 - Discontinuous Gel Electrophoresis, Protein Mobilities and Apparent Size Determination
Process: Discontinuous Gel Electrophoresis & Protein Size Determination
Lab 8.1 - Discontinuous SDS Gel Electrophoresis
Unit 9
Lecture 9 - Immunochemical Techniques
Innov.: Immunochemical Techniques
Innov.: The Enzyme Linked Immunosorbent Assay (ELISA)
Innov.: How the Immune System Learns About Self
Innov.: Making Monoclonal Antibodies That Won't Fight Back
Lab 9.1 - Western Blotting
Process: Immunoblotting
Process: Western Blots Using Stained Protein Gels
Unit 10
Lecture 10 - Combinatorial Biochemical Technology
Innov.: Examples of Combinatorial Techniques
Innov.: Making Antibody Fragments Using Phage Display Libraries
Innov.: Building a Better Enzyme
Innov.: The ImmunoZAP Cloning and Expression System
Part 1 Terms List
Part 2 Terms List
Laboratory Reagents
Abbreviations List
Copyright Acknowledgements
Suggested Schedule
Supplies Required
Innov., Innovation/Insight
Theory, Theory/Principles
Process, Process Rationale
Vend. Lit., Vendor Literature
Alt. App., Alternative Approach

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