A Flexible System of Enzymatic Analysis - 1st Edition - ISBN: 9780124579507, 9780323156714

A Flexible System of Enzymatic Analysis

1st Edition

Authors: Oliver Lowry
eBook ISBN: 9780323156714
Imprint: Academic Press
Published Date: 28th August 1972
Page Count: 304
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Description

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.

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

Enzyme Kinetics

Chapter 3 Glassware

Constriction Pipettes

Tubes

Chapter 4 Typical Fluorometric Procedures for Metabolic Assays

A One-Step Metabolite Assay with TPN+

A One-Step Metabolite Assay with DPNH

A Multistep Metabolite Assay (for Inorganic Phosphate)

Chapter 5 Measurement of Enzyme Activities with Pyridine Nucleotides

A One-Step Enzyme Assay with TPN+ (Isocitric Dehydrogenase)

A One-Step Enzyme Assay with DPNH (Glycero-P Dehydrogenase)

A Multistep Enzyme Assay (P-Glucomutase)

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

ATP and P-Creatine

Citrate

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

Acid-Base Made Easy

Statistical Shortcuts

Construction of Dissecting Knives

Microtome Knife Sharpening

Table of Indicators

Preparation and Storage of Common Stock Solutions

Normality of Concentrated Acids and Bases

Commercial Sources of Equipment and Supplies

Abbreviated List of Atomic Weights

References

Subject Index






Details

No. of pages:
304
Language:
English
Copyright:
© Academic Press 1972
Published:
Imprint:
Academic Press
eBook ISBN:
9780323156714

About the Author

Oliver Lowry