Thomas E. Crowley, Jack Kyte

Experiments in the Purification and Characterization of Enzymes (eBook, ePUB)

A Laboratory Manual

• Format: ePub

Produktbeschreibung

Experiments in the Purification and Characterization of Enzymes: A Laboratory Manual provides students with a working knowledge of the fundamental and advanced techniques of experimental biochemistry. Included are instructions and experiments that involve purification and characterization of enzymes from various source materials, giving students excellent experience in kinetics analysis and data analysis. Additionally, this lab manual covers how to evaluate and effectively use scientific data. By focusing on the relationship between structure and function in enzymes, Experiments in the Purification and Characterization of Enzymes: A Laboratory Manual provides a strong research foundation for students enrolled in a biochemistry lab course by outlining how to evaluate and effectively use scientific data in addition to offering students a more hands-on approach with exercises that encourage them to think deeply about the content and to design their own experiments. Instructors will find this book useful because the modular nature of the lab exercises allows them to apply the exercises to any set of proteins and incorporate the exercises into their courses as they see fit, allowing for greater flexibility in the use of the material.

Written in a logical, easy-to-understand manner, Experiments in the Purification and Characterization of Enzymes: A Laboratory Manual is an indispensable resource for both students and instructors in the fields of biochemistry, molecular biology, chemistry, pharmaceutical chemistry, and related molecular life sciences such as cell biology, neurosciences, and genetics.

• Offers project lab formats for students that closely simulate original research projects
• Provides instructional guidance for students to design their own experiments
• Includes advanced analytical techniques
• Contains adaptable modular exercises that allow for the study proteins other than FNR, LuxG and LDH
• Includes access to a website with additional resources for instructors

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Produktdetails

• Verlag: Elsevier Science & Techn.
• Seitenzahl: 266
• Erscheinungstermin: 11. Januar 2014
• Englisch
• ISBN-13: 9780124095939
• Artikelnr.: 40425443

Autorenporträt

Thomas Crowley studied biochemistry as an undergraduate at the University of Illinois Urbana-Champaign. He then pursued graduate studies in molecular biology in the Division of Biology at the California Institute of Technology. As a graduate student and later during postdoctoral studies he used biochemical and genetic methods to examine the regulation of gene expression and the intracellular localization of proteins during animal development. He has taught courses covering a wide range of chemical and biological topics such as general chemistry, biochemistry, microbiology, cell biology and developmental biology. These courses were taught at Montclair State University in New Jersey, Columbia University in New York City, the University of California San Diego and National University in La Jolla, California. He has authored articles derived from his research in a variety of journals and articles derived from his teaching in Biochemistry and Molecular Biology Education (BAMBED). He is currently an adjunct faculty member in the Department of Mathematics and Natural Sciences at National University and a member of the American Chemical Society.

Inhaltsangabe

Safety Guidelines for Biochemistry LaboratoriesGeneral Guidelines for
Handling Solutions of ProteinMaintaining a Laboratory NotebookIntroduction
to Enzymes Catalyzing Oxidation-Reductions with the Coenzyme
NAD(P)Computational Techniques for Biochemistry Software for Analysis of
DataComputational Methods and Bioinformatics for Studying the Structure and
Function of ProteinsBackground: Software and Databases; 1. Retrieve Entries
from a Database and Compare Sequences of Amino Acids; 2. Compare the
Tertiary Structures of PolypeptidesSection 1: FNR; Purification and
Characterization of Ferredoxin-NADP+ Reductase from Chloroplasts of S.
oleracea1. Preparation of a Lysate of Chloroplasts and Sequestration of
Proteins by an Anion-exchange Solid Phase; 2. Elution of Proteins from the
Anion-exchange Solid Phase and Assays for the Concentration of Total
Protein and FNR Activity; 3. Isolation of FNR by Adsorption and Elution4.
Electrophoresis of Proteins from the Chloroplasts and Purified FNR on Gels
of Polyacrylamide Cast in Solutions of Dodecyl Sulfate; 5. Kinetic Assay
for the Km of FNR for NADPH; 6. Estimation of the Size and Quaternary
Structure of Native FNR Using Chromatography by Molecular Exclusion; 7.
Detection of FNR in Extracts from Chloroplasts of Spinach by Immunoblotting
Section 2: LuxG; Purification and Characterization of a Recombinant FMN
Reductase from P. leiognathi1. Expression of LuxG-hexahistidine in E. coli
and Its Release from the Cells by Lysis; 2. Affinity Adsorption of
LuxG-hexahistidine to a Solid Phase to which Ni2+ is Chelated; 3.
Electrophoresis of the Polypeptides in the Lysate and Purified
LuxG-hexahistidine; 4. Assay for the Flavin Reductase Activity of
LuxG-hexahistidine; 5. Determination of the Michaelis Constant of
LuxG-hexahistidine for NADH; 6. Determination of the Molar Concentration of
LuxG-hexahistidine SpectrophotometricallySection 3: LDH; Purification and
Characterization of Bovine LDH1. Preparation of the Isoenzymes of LDH from
Extracts of Various Tissues; 2. Spectrophotometric Assay of the Activity of
Bovine LDH; 3. Separation and Visualization of the Isoenzymes of LDH by
Electrophoresis on Cellulose Acetate; 4. Fractionation of Proteins in
Bovine Heart by Precipitation with Sulfate; 5. Purification of LDH from
Heart by Affinity Adsorption and Elution; 6. Concentration of Protein by
the Bradford Stain and Absorbance at 280 nm; 7. Electrophoresis of Native
Proteins from Bovine Heart and Purified LDH on a Gel of Polyacrylamide; 8.
Electrophoresis of Unfolded Polypeptides from Bovine Heart and Purified LDH
Saturated with Dodecyl Sulfate; 9. Determination of the Michaelis Constant,
KmNADH, of the Isoenzymes of LDH for NADH; 10. Chromatography by Molecular
Exclusion to Evaluate the Quaternary Structure of LDH; 11. Detection of LDH
in Extracts of Bovine Tissue by ImmunoblottingSection 4: Experimental
Design; 1. Association of FNR with the Membranes of the Thylakoids in the
Chloroplasts from Spinach; 2. Search for Multiple Flavin Reductases in P.
leiognathi; 3. Assay for LDH Activity Specific to (R)-lactate in Bovine
MitochondriaAppendices I. Measurement of Absorbance with Multiwell Plates
and an Automated Plate Reader; II. Operation of the Spectronic 20D+
Spectrophotometer