Description
Experiments in the Purification and Characterization of Enzymes
A Laboratory Manual
Authors: Crowley Thomas E., Kyte Jack
Language: EnglishSubject for Experiments in the Purification and Characterization of...:
Keywords
Michaelis constant; aerobic catabolism; affinity purification; anaerobic catabolism; anion exchange; bacterial luminescence; bioluminescence; chloroplast; d-lactate; enzyme kinetics; experimental design; flavin mononucleotide reductase; immobilized metal ion affinity purification; immunoblot; isoenzymes; l-lactate; membrane association; mitochondrion; molecular exclusion chromatography; oxidation-reduction; photosynthesis; quaternary structure; recombinant expression; stereoisomer-specific enzymes; student-directed; subcellular fractionation; tetramer; thylakoid; tissue-specific expression; nicotinic adenine dinucleotide (NAD); ferredoxin; flavin mononucleotide; l-lactate; pyruvate; reduction potential; photosynthesis; bacterial luminescence; aerobic catabolism; anaerobic catabolism; active site; Michaelis constant; quaternary structure; thylakoid; chloroplast; spreadsheet software; bioinformatics software; computational biochemistry; protein sequence database; amino acid sequence comparison; tertiary structure comparison; nucleotide seqeunce database; nucleotide sequence comparison; molecular evolution
266 p. · 21.4x27.6 cm · Paperback
Description
/li>Contents
/li>Readership
/li>Biography
/li>Comment
/li>
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.
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 ImmunoblottingSection 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
Jack Kyte graduated from Carleton College in 1967, completed his doctoral studies at Harvard University in the laboratory of Guido Guidotti in 1972, and was a postdoctoral fellow with S. Jonathan Singer at the University of California at San Diego. In 1974 he joined the faculty of the Department of Chemistry at the University of California at San Diego and retired from the department as Professor of Chemistry in 1999. In his research, he studied the structures of Na+/K+-exchanging ATPase and anion carrier using chemical labeling and site-directed immunochemistry. He also studied the mechanism of the activation of the receptor protein-tyrosine kinase for epidermal growth factor and the mechanism of the reaction catalyzed by ribonucleoside-diphosphate reductase. He has written two books, Structure in Protein Chemistry and Mechanis
- 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
These books may interest you
Techniques in Molecular Medicine 105.49 €