Micromethods in Molecular Biology

This book is based on practical experience and is therefore written as a prac­ tical manual. The fore-runners of the book were the manuals of the first and second EMBO-Courses on "Micromethods in Molecular Biology" which were held in G6ttingen in the spring of 1970 and the autumn of 1971. This book may serve as a manual not only for the participants of the third EMBO-Course to be held in G6ttingen in autumn 1973, but also for all experimenters who are interested in using micromethods. It must be emphasized from the outset that this book is conceived as a "cook book" and not as a monograph which attempts to revue the literature on micromethods critically. The methods described here in detail are performed routinely in the authors' laboratories and include all the practical details necessary for the successful appli­ cation of the micromethods. There are many other sensitive and excellent micro­ methods which are not included in this book, because the authors feel that in a "cook book" only methods for which they have personal experience and profi­ ciency should be described. Some readers may feel that the title promises more than the present contents of this book; however, if sufficient interest is shown in this volume, it may be possible to remedy such deficiencies in future editions.

Contenu

1 Micro-Electrophoresis on Polyacrylamide Gels.- Micro-Disc Electrophoresis.- Use of Capillaries.- Preparation of Gels.- Electrophoresis.- Isolation of Individual Protein Fractions.- Microanalysis of DNA-Dependent DNA-Polymerase.- Binding Test.- Polymerisation Test.- Microanalysis of DNA-Dependent RNA-Polymerase.- Quantitative Assay for Dehydrogenases.- Assay Conditions.- Enzyme Kinetics.- Michaelis-Menten Kinetics and the Determination of Enzyme Activity.- Micro Isoelectric Focusing.- Micro-Electrophoresis on Gradient Gels.- Preparation of Gradient Gels.- Protein Fractionation.- Fractionation of RNA.- Micro-Electrophoresis in SDS Gradient Gels.- Autoradiography of Microgels.- Sources of Error in Gradient Gel Electrophoresis.- Concluding Remarks.- Literature.- 2 Micro-Determination of Amino Acids and Related Compounds with Dansyl Chloride.- Dansylation Reaction.- Preparation of the Stock Solution of Dansyl Chloride.- Dependence of the Dansyl Reaction on pH.- Dependence of the Dansyl Reaction on the Incubation Time.- Practical Procedure.- Dansyl Reaction in Micro Scale.- Microchromatography.- Evaluation of Microchromatograms.- Evaluation by Autoradiography.- Identification of Dansyl Derivatives.- Characterization of tRNA.- Determination of C- and N-Terminal Amino Acids.- Determination of Amino Acids from Biological Material.- Determination of Mono-Amines.- Concluding Remarks.- Literature.- 3 Micro-Determination of Phospholipids.- General Remarks.- Calibration Curves.- Analysis of Biological Material.- Extraction of Phospholipids.- Micro-Chromatography.- Two-Dimensional Micro-Chromatography.- One-Dimensional Micro-Chromatography.- Two-Dimensional, Two-Step, Technique for Micro-Chromatography of Lipids.- Biological Applications.- Literature.- 4 Micro-Diffusion Techniques.- Two-Dimensional Micro-Immunodiffusion.- Characterization of a Polymerase-Template Complex by Two-Dimensional Micro-Diffusion.- Radial Micro-Diffusion.- Micro Antigen-Antibody Crossed Electrophoresis.- One-Dimensional Multi-Stage Micro-Immunoelectrophoresis.- Literature.- 5 Capillary Centrifugation.- Preparative Capillary Centrifugation.- Analytical Capillary Centrifugation.- Literature.- 6 Micro-Electrophoresis for RNA and DNA Base Analysis.- Biological Material.- Preparation of Microinstruments and Their Use.- Procedure for the Measurement of a Volume.- Extraction of Nucleic Acids.- Medium for Electrophoresis.- Buffers for Electrophoresis.- Micro-Electrophoresis (Microphoresis).- Photographic and Photometric Measurements.- RNA Analysis.- DNA Analysis.- Determination of Total Amounts of Nucleic Acids.- Biological Applications.- Literature.- 7 Determination of the Dry Mass of Small Biological Objects by Quantitative Electron Microscopy.- Prerequisites.- Electron Microscope.- Accelerating Voltage.- Contrast Aperture.- Exposure System.- Darkroom.- Densitometry.- The Principle of the Method of Dry Mass Determination with the Electron Microscope.- Estimation of the Useful Mass Range of an Electron Microscope.- Upper and Lower-Limits.- Consideration of the Photographic Fog.- Measurement of Transmission.- From Sample to Result.- Electron Microscopy.- Measurements with the IPM-2.- Calculation of Mass.- Standardization.- Single-Line Scanning Measurements.- Concentration of Solids.- Preparing an Object for Quantitative Electron Microscopy.- Simple Preparatory Steps.- Preparing Human Chromosomes.- Chromosome Culture.- Preparation of FORMVAR-Coated Grids.- Dehydration and Critical-Point Drying.- Summary.- Literature.- 8 The Construction and Use of Quartz Fiber Fish Pole Balances.- Optical Measurements.- The Choice and Manufacture of Suitable Quartz Fibers.- Making the Fiber.- Balance Cases and Final Assembly.- Mounting the Balance.- Weighing.- Calibration.- Weight Correction for Adsorption of Gases and Moisture in Tissues.- 9 Microphotometry.- Preface.- Photometry.- Photometers.- Microphotometry.- Microphotometers.- Spot Measurements.- Line Scanning.- Area Scanning.- Addendum.- Remarks Regarding the Microphotometry of Autoradiographs.- Literature.- 10 Cytofluorometry.- Description of Instrument.- Basic Equipment.- Light Sources.- Filters and Reflectors.- Objectives.- Fluorescence Standard.- Microscope Photometer.- Recording of the Intensity of Fluorescence.- Use of Instrument.- Adjustment of the Instrument.- Measuring Procedure.- Some Applications.- Material.- Fixation.- Storage.- Staining Reactions.- DNA.- Histones.- Total Protein.- Arginine.- Lysine.- Sulfhydryl Groups.- Successive Measurements.- Analysis and Evaluation of Data.- Literature.- 11 Quantitative Autoradiography at the Cellular Level.- Biochemical Aspects.- The Precursor Pool.- Turnover of the End-Product.- Reutilization of the Labeled Compounds.- The Nuclear Emulsion.- Response to ?-Rays.- Development.- Fading of the Latent Image.- The Autoradiograph.- Track Autoradiography.- Grain-Density Autoradiography.- ?-Self-Absorption and ?-Absorption.- Back-Scattering.- Geometric Factors.- Chemography.- Background.- Preparing the Samples.- Fixation.- Autoradiography of Soluble Compounds.- Quantitative Determination of Substances.- Track Counting.- Grain Yield.- Radioactive Standard Sources.- Manual and Automatic Methods of Grain Counting.- Visual Counting.- Silver Grain Photometry.- Application. The Rate of Synthesis of DNA in Individual Cells.- Literature.- 12 Micro-Dialysis.- Micro-Dialysis Chamber.- Micro-Electrodialysis.- Equilibrium Dialysis.- Literature.- 13 Micro-Homogenisation.- Literature.- 14 Wet Weight Determination in the Lower Milligram Range.- Literature.- 15 Micro-Magnetic Stirrer.- 16 Production of Capillary Pipettes.- Literature.