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Cover

Cloning, Gene Expression, and Protein Purification

Experimental Procedures and Process Rationale

Charles Hardin, Jennifer Edwards, Andrew Riell, David Presutti, William Miller, and Dominique Robertson

Publication Date - March 2001

ISBN: 9780195132946

448 pages
Paperback
8-1/2 x 11 inches

In Stock

Retail Price to Students: $99.95

Description

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

About the Author(s)

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
    Appendices
    Part 1 Terms List
    Part 2 Terms List
    Laboratory Reagents
    Abbreviations List
    Copyright Acknowledgements
    Suggested Schedule
    Supplies Required
    Index
    (Abbreviations:
    Innov., Innovation/Insight
    Theory, Theory/Principles
    Process, Process Rationale
    Vend. Lit., Vendor Literature
    Alt. App., Alternative Approach