Methods of Protein and Nucleic Acid Research: Volume 1: Electrophoresis - Isoelectric Focusing Ultracentrifugation

ISBN-13: 9783642874871 / Angielski / Miękka / 2012 / 344 str.

L. a. Osterman

Opis

Electrophoresis is the leading method among those used in the investigation of pro teins and nucleic acids. A paper on the study of these biopolymers without recourse to electrophoresis at each fractionation or characterization step is very unlikely to be encountered in the current scientific literature. This method enables separation of macromolecules according to characteristic features such as size (or molecular weight), shape, secondary structure and electric charge and these parameters can in fluence electrophoretic properties either separately or jointly. The physical background of this method is as follows. Macromolecules in a buf fer solution may become charged; the sign and magnitude of the total electrical charge depending on the pH of the medium. When such a solution is put into an insulated channel, e.g. in a glass tube, and a voltage applied at the ends of the chan nel, an electric field will be formed and direct current will pass through solution. The field strength can be defined as the potential difference at the ends of the chan nel (or its section) with respect to its length (v/cm). When exposed to an electrical field macromolecules will migrate towards the cathode or the anode according to their net charge and frictional force will limit the migration velocity."

Spis treści

I Electrophoresis.- 1 Gels for Electrophoresis.- 1.1 Polyacrylamide Gel (PAAG).- 1.1.1 Reagents.- 1.1.2 Polymerization Process.- 1.1.3 Selection of Concentration of Monomers.- 1.2 Agarose.- 1.3 Mixed Gels (Agarose + PAAG).- 1.4 Cellulose Acetate Strips Impregnated with PAAG.- 2 Techniques of Gel Preparation and Necessary Equipment.- 2.1 Vertical Tube Electrophoresis.- 2.2 Vertical Slab Electrophoresis.- 2.3 Horizontal Slab Electrophoresis.- 3 Electrophoresis of Proteins.- 3.1 General Remarks.- 3.1.1 Migration of Proteins in Gels.- 3.1.2 Electric Field Strength.- 3.1.3 Choice of Running Gel Buffer.- 3.1.4 Heat Evolution During Electrophoresis.- 3.1.5 Gel Loading: Width of Protein Bands.- 3.1.6 Introduction of Urea and ?-Mercaptoethanol. Possible Artifacts.- 3.1.7 Tracking Dyes.- 3.1.8 Separation of Proteins According to Size and Charge.- 3.1.9 Choice of Running Buffer.- 3.1.10 Use of Urea.- 3.1.11 Gel Loading and Sample Preparation.- 3.1.12 Selected Examples.- 3.2 Separation of Proteins on the Basis of Size in the Presence of Sodium Dodecyl Sulphate (SDS).- 3.2.1 Principles of the Method.- 3.2.2 Selection of Gel Porosity.- 3.2.3 Influence of Urea and Nonionic Detergents.- 3.2.4 Selection of Running Buffer.- 3.2.5 Preparation of Protein Samples.- 3.2.6 Electrophoresis.- 3.2.7 Staining and Elution of Proteins.- 3.3 Disc Electrophoresis.- 3.4 Pore Gradient PAAG.- 3.5 Two-Dimensional PAAG-Electrophoresis.- 3.6 Electrophoresis in the Presence of Triton X-100 and Cetavlon.- 3.6.1 Triton X-100.- 3.6.2 Cetavlon.- 3.6.3 Affinity Electrophoresis.- 3.7 Staining of Proteins in PAAG.- 3.7.1 Acidic Dyes.- 3.7.2 Other Dyes and Methods of Staining.- 3.7.3 Fluorescent Dyes.- 3.7.4 Detection of Enzymes After Electrophoresis.- 3.7.5 Detection of Protein Bands by Precipitation of SDS.- 3.8 Elution of Proteins from the Gel.- 3.9 Determination of Protein Radioactivity After PAGE.- 3.9.1 Counting of Protein Eluates.- 3.9.2 Solubilization of PAAG.- 3.9.3 Gels Impregnated with Scintillant.- 3.9.4 Autoradiography.- 3.9.5 Fluorography.- 3.10 Preparative Electrophoresis of Proteins.- 4 Electrophoresis of Nucleic Acids.- 4.1 General Remarks.- 4.1.1 Choice of Buffer and Electric Field Strength.- 4.1.2 Choice of Character and Porosity of Gel.- 4.1.3 Influence of Secondary Structure of Nucleic Acids.- 4.1.4 Marker Molecules.- 4.1.5 Tracking Dyes.- 4.2 Electrophoresis Under Mild Conditions.- 4.2.1 Fractionation of Plasmids and DNA Fragments.- 4.2.2 Fractionation of RNA.- 4.3 Electrophoresis in Denaturating Gels.- 4.3.1 Alkaline Gels.- 4.3.2 Urea-containing Gels. Sequencing of DNA and RNA.- 4.3.3 Denaturating Gels Containing Formamide.- 4.3.4 Denaturating Gels Containing Formaldehyde.- 4.3.5 Gels Containing Methylmercury Hydroxide.- 4.3.6 Denaturation of Nucleic Acids with Glyoxal.- 4.4 Application of Pore Gradient PAAG.- 4.5 Two-Dimensional Electrophoresis of Nucleic Acids and Their Fragments.- 4.6 Specific Examples of Electrophoresis of Nucleic Acids.- 4.6.1 Electrophoresis of the Ternary Complex of DNA-RNA-polymerase-RNA in Agarose Gel.- 4.6.2 Detection of mRNA After Agarose-Gel Electrophoresis of Total RNA.- 4.6.3 The Use of Liquid (Non-Cross-Linked) PAAG.- 4.6.4 Separation of Okazaki Fragments from a Total Cell Lysate.- 4.7 Staining of Nucleic Acids After Electrophoresis.- 4.8 Elution of Nucleic Acids from Gels.- 4.8.1 Elution from PAAG Due to Diffusion.- 4.8.2 Elution from Agarose Gels.- 4.8.3 Electrophoretic Elution.- 4.8.4 Transfer of Nucleic Acids from Gels onto Nitrocellulose Filters and Diazopaper.- 4.9 Determination of Radioactivity.- 4.10 Preparative Electrophoresis of Nucleic Acids.- II Isoelectric Focusing (IEF).- 1 Principle of the Method.- 1.1 Migration of Proteins in a pH Gradient.- 1.2 Ampholytes for IEF.- 1.3 Generation of a pH Gradient.- 1.4 Electrical Conductivity Problem.- 1.5 Comparison of Properties of Commercial Ampholytes.- 1.6 Danger of Appearance of a Zone of Pure Water.- 1.7 Resolving Power. Choice of pH Interval.- 1.8 Physicochemical Characteristics of Ampholytes.- 1.9 Endosmosis.- 1.10 Immobilized pH Gradients.- 2 Analytical Variations of IEF.- 2.1 IEF in Horizontal Slabs of PAAG.- 2.2 Preparation of PAAG for IEF.- 2.3 Use of Urea and Detergents.- 2.4 Preparation of Slabs and Apparatus.- 2.5 Sample Application.- 2.6 Operating Conditions and Duration of IEF.- 2.7 Measurement of pH.- 2.8 Staining of Protein Zones.- 2.8.1 Two-Step Staining.- 2.8.2 Enzymatic Reactions.- 2.9 IEF in Tubes of PAAG.- 2.10 IEF in Horizontal Slabs of Agarose.- 3 Two-Dimensional Fractionation of Proteins.- 3.1 Method of O’Farrell.- 3.2 Modifications of the Method of O’Farrell.- 3.3 Sparingly Soluble Membrane Proteins.- 3.4 Electrophoresis Together with the pH Gradient Generation.- 3.5 Interpretation of Two-Dimensional Fractionation Patterns of Proteins According to O’Farrell.- 3.6 The Use of IEF for the Second-Dimension Run.- 3.7 Electrophoresis of Proteins Across the pH Gradient.- 4 Preparative Variants of IEF.- 4.1 IEF in a Layer of Granulated Gel.- 4.1.1 Preparation of the Gel Bed.- 4.1.2 Sample Application.- 4.1.3 Conditions of IEF.- 4.1.4 Detection and Elution of Proteins.- 4.2 IEF in Sucrose Density Gradients.- 4.2.1 Design of the Column.- 4.2.2 Filling of the Column.- 4.2.3 Sample Application.- 4.2.4 Operating Conditions of IEF.- 4.2.5 Unloading of the Column.- 4.2.6 Specific Problems.- 4.2.7 Choice of Electrode Polarity.- 4.2.8 Danger of Precipitation.- 4.2.9 Solubility Problems.- 4.2.10 Maximum Loading.- 4.2.11 Fractionation of Ampholytes into Narrow pH Intervals.- 4.2.12 The ISCO Columns.- 5 Isotachophoresis.- III Ultracentrifugation.- 1 Sedimentation — Basic Concepts.- 1.1 Buoyant Density of Particles.- 2 Ultracentrifuge.- 2.1 Principles of Action and Arrangement of Main Units.- 2.2 Points to be Checked by Experimenter.- 2.3 Making a Run.- 3 Rotors and Tubes.- 3.1 Fixed Angle Rotors.- 3.1.1 Tubes for Fixed Angle Rotors.- 3.1.2 Care of Rotors and Tubes.- 3.2 Swinging Bucket Rotors.- 3.3 Vertical Tube Rotors.- 3.4 Maximum Speed of Rotation.- 3.5 Zonal Rotors.- 3.5.1 Specific Requirements in the Exploitation and Care of Zonal Rotors.- 4 Differential Centrifugation (Pelleting).- 5 Rate-Zonal Centrifugation.- 5.1 Sedimentation Constant.- 5.2 Isokinetic Density Gradient.- 5.3 Choice of Medium for Rate-Zonal Centrifugation.- 5.4 Properties of Sucrose Solutions.- 5.5 The Shape of the Sucrose Gradient.- 5.6 Preparing and Unloading Sucrose Gradients.- 5.6.1 Loading of the Sample onto a Gradient.- 5.6.2 Fractionation of the Gradient.- 5.6.3 Analysis of Gradients.- 5.7 Choice of Operating Conditions.- 5.8 Examples of Centrifugation in Sucrose Gradients.- 5.8.1 Fractionation of Ribonucleoproteins.- 5.8.2 Fractionation of RNA.- 5.8.3 Fractionation of DNA.- 6 Equilibrium (Isopycnic) Centrifugation.- 6.1 Modes of Preparation and General Characterization of Density Gradients.- 6.2 Choice of Salt for Preparation of Density Gradients.- 6.3 Density Gradients of CsCl Solutions.- 6.3.1 Preformed Gradients.- 6.3.2 Equilibrium Centrifugation in Fixed Angle Rotors.- 6.3.3 Centrifugation in Vertical Tube Rotors.- 6.3.4 Unloading and Monitoring of Isopycnic Gradients.- 6.3.5 Examples of Equilibrium Centrifugation in Density Gradients of CsCl.- 6.4 Cs2SO4 Density Gradients.- 6.5 Caesium and Rubidium Trichloroacetate Density Gradients.- 6.6 KI and NaI Density Gradients.- 6.7 Metrizamide Density Gradients.- 7 Centrifugation in Zonal Rotors.- References to Part I.- References to Part II.- References to Part III.- List of Selected Examples.- Nomograms for Calculation of Centrifugation Time (for Practical Use).