Keywords:
Enzymatic analysis -- Laboratory manuals.
;
Electronic books.
Description / Table of Contents:
A Flexible System of Enzymatic Analysis is a multipurpose manual of laboratory methods that offers a systematic scheme for the analysis of biological materials from the level of the wholeorgan down to the single cell and beyond. It intends to guide the development of methods, the refinement of old ones, and the adaptation in general of methods to almost any scale of sensitivity. This manual is organized into three parts: a general section, one on quantitative histochemistry, and an appendix containing information that may be useful to have at the bench. The general section is comprised of nine chapters that focus on properties of the pyridine nucleotides, kinetics, and glassware, as well as on improvement, modification, adaptation, trouble shooting, and development of methods. This part also describes the preparation of tissues for analysis, the enzymatic cycling methods, and a compendium of 36 metabolite assays. The quantitative histochemistry section is comprised of four chapters that include information on the preparation of frozen-dried material and dissection of samples for analysis; the fishpole balance for weighing samples; and the generalities of analysis with emphasis on the ""oil well technique."" This book will be useful to novices as well as experts who are familiar with other analytical styles.
Type of Medium:
Online Resource
Pages:
1 online resource (304 pages)
Edition:
1st ed.
ISBN:
9780323156714
URL:
https://ebookcentral.proquest.com/lib/geomar/detail.action?docID=1172935
DDC:
574.1/9285
Language:
English
Note:
Front Cover -- A Flexible System of Enzymatic Analysis -- Copyright Page -- Table of Contents -- PREFACE -- Part I: GENERAL PRINCIPLES -- Chapter 1. Pyridine Nucleotides -- Spectral Absorption and Fluorescence of Reduced Pyridine Nucleotides -- Limitations of Fluorometry -- Fluorescence Reference Standards -- Excitation and Emission Wavelengths and Light Filters -- Conversion of Pyridine Nucleotides in Alkali into Fluorescent Products -- Increased Fluorescence from DPNH and TPNH with Strong Alkali -- Stability and Selective Destruction of Reduced and Oxidized Forms of Pyridine Nucleotides -- Destruction of DPNH and TPNH in Acid and Stability in Alkali -- Destruction of DPN+ and TPN+ in Alkali and Stability in Acid -- Preparation, Standardization, and Storage of Pyridine Nucleotide Solutions -- Fluorometers -- Chapter 2. A Kinetic Primer for the Tissue Analyst -- NONENZYME KINETICS -- First-Order Reactions -- Second-Order Reactions -- ENZYME KINETICS -- One-Step Reactions with One Substrate -- First-Order Enzyme Reactions -- Mixed Zero-Order and First-Order Enzyme Reactions -- One-Step Reactions with Two Substrates -- Enzyme Reactions with Second-Order Kinetics -- Two-Step Reactions -- Other Factors Affecting Enzyme Kinetics -- Chapter 3. Glassware -- CONSTRICTION PIPETTES -- The Role of Surface Tension on Pipetting -- Effect of Changes in Surface Tension and Viscosity -- Pipetting Technique -- Special Considerations with Small Pipettes and Small Tubes -- Transferring the Whole Sample (" Total Transfer ") -- Common Pipetting Errors -- Rinsing Pipettes -- Making Constriction Pipettes -- Calibration of Pipettes -- Cleaning and Storage of Constriction Pipettes -- TUBES -- Fluorometer Tubes -- Tubes for 25-200 μl Volumes -- Tubes for Volumes of Less than 25 μl -- How to Make Micro Test Tubes -- Cleaning and Storage of Tubes.
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Chapter 4. Typical Fluorometric Procedures for Metabolic Assays -- A ONE-STEP METABOLITE ASSAY WITH TPN+ -- Spectrophotometric Procedure -- Five Fluorometric Procedures for the Measurement of Glucose-6-P -- A ONE-STEP METABOLITE ASSAY WITH DPNH -- Spectrophotometric Procedure -- Four Fluorometric Procedures for the Measurement of α -ketoglutarate -- A MULTISTEP METABOLITE ASSAY (FOR INORGANIC PHOSPHATE) -- Spectrophotometric Procedure -- Four Fluorometric Procedures for the Measurement of Pi -- Chapter 5. Measurement of Enzyme Activities with Pyridine Nucleotides -- A ONE-STEP ENZYME ASSAY WITH TPN+ (ISOCITRIC DEHYDROGENASE) -- Spectrophotometric Procedure -- Three Fluorometric Procedures -- A ONE-STEP ENZYME ASSAY WITH DPNH (GLYCERO-P DEHYDROGENASE) -- Spectrophotometric Procedure -- Three Fluorometric Procedures -- A MULTISTEP ENZYME ASSAY (P-GLUCOMUTASE) -- Spectrophotometric Procedure -- Three Fluorometric Procedures -- Chapter 6. Improvement, Modification, Adaptation, Trouble Shooting, and Development of New Methods -- Simplification and Improvement -- Adaptation of Pyridine Nucleotide Methods to Increase Sensitivity -- Development of a New Method -- Chapter 7. Preparation of Tissues for Analysis -- Preparation for Enzyme Assays -- Preparation for Metabolite Analyses -- Preparation of Tissue Extracts -- The Use of Methanol-HCl in Preparing Extracts -- Fluorescence of Tissue Extracts -- Measurement of Pyridine Nucleotides in Tissues -- Chapter 8. Enzymatic Cycling -- TPN Cycle -- DPN Cycle -- ATP-ADP Cycle -- Glutamate- α-ketoglutarate Cycle -- Other Cycles -- Double Cycling -- Chapter 9. A Collection of Metabolite Assays -- ADP and AMP -- ALANINE -- ASPARTATE -- ATP and P-Creatine -- CITRATE -- Creatine -- Dihydroxyacetonephosphate: Method I (with Glycero-P-dehydrogenase) -- Fructose -- Fructose-6-phosphate.
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Fructose-1, 6-diphosphate: Method I -- Fructose-1,6-diphosphate (Method II) Dihydroxyacetonephosphate (Method II), and Glyceraldehydephosphate -- Fumarate -- Glucose -- Glucose-1 -phosphate -- Glucose-6-phosphate -- Glucose-1,6-diphosphate -- Glutamate -- α-Glycerophosphate: Method I -- α-Glycerophosphate: Method II -- Glycogen -- Isocitrate -- Lactate: Method I -- Lactate: Method II -- Malate: Method I -- Malate: Method II -- Oxalacetate -- 6-P-Gluconate -- 3-P-Glycerate: Method I -- 3-P-Glycerate: Method II -- P-Pyruvate -- Pyruvate -- Total Nucleotide Triphosphates -- Uridine-5-diphosphoglucose -- Part II: QUANTITATIVE HISTOCHEMISTRY -- Introduction -- Chapter 10. Preparation of Tissues And Sections -- Freezing -- Storage of Frozen Tissues -- Mounting the Tissue for Cutting -- Sectioning the Tissue -- Drying of Samples -- Storage of Dry Sections -- Chapter 11. Dissection and Histological Control -- Dissection and Balance Room -- Removal of Sections from Evacuation Tube -- Dissection -- Handling Dissected Samples -- Identification and Histological Control -- Sample Carriers -- Defatting Frozen Dried Samples -- Sample Volume -- Chapter 12. The Quartz Fiber Fishpole Balance -- Measurement of Displacement and Useful Range -- Choice of Suitable Fiber. Sensitivity as a Function of Length and Diameter of the Fiber -- Balance Fibers -- Pans -- Balance Case -- Mounting the Fiber -- Lighting and Viewing -- Weighing -- Correction of Weight for Adsorption of Gases and Moisture -- Calibration of Balances and Testing Linearity -- Cleaning the Balance Fiber -- Chapter 13. Histochemical Analysis -- Introduction -- Enzyme Stability at High Dilution -- Addition of Sample and Reagent -- Oil Well Technique -- Reagent Wells -- Pipettes -- Microscope and Working Stage -- Addition of Sample and Reagent -- Special Considerations -- Part III: APPENDIX -- Appendix.
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Acid-Base Made Easy -- Statistical Shortcuts -- Construction of Dissecting Knives -- Microtome Knife Sharpening -- Table of Indicators -- Preparation and Storage of Common Stock Solutions -- Normality o f Concentrated Acids and Bases -- Commercial Sources of Equipment and Supplies -- Abbreviated List of Atomic Weights -- References -- SUBJECT INDEX.
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