Basic Methods for the Biochemical Lab (Springer Lab Manuals)

Basic Methods for the Biochemical Lab elucidates proven lab procedures and practical hints for research in analytical and preparative biochemistry, as well as summarizing key data in numerous tables. To further emphasize the practical aspects and to minimize the text, theoretical introductions into the methods are mostly omitted. The chapters cover: quantitative methods (proteins, nucleic acids, phospholipids and carbohydrates), electrophoresis (several polyacrylamide and agarose systems, 2D-PAGE, detection methods and affinity electrophoresis), chromatographic protocols (thin-layer chromatography, GPC, IEC, affinity chromatography, HPLC), immunochemical protocols (hapten-carrier and enzyme conjugation, immunization and antibody purification, immune affinity chromatography, ELISA), centrifugation (differential and density gradient centrifugation for cells and cellular fractions), radioactivity (labeling and counting), buffers (buffer properties and compositions). In additional chapters, tables compile a broad array of useful information, e.g. SI units conversion factor, detergent, protein and nucleotide data, and the basic principles of statistics and enzyme and receptor kinetics are reviewed. This first English-language edition of a successful German-language manual will be a valuable resource for students and experts in biochemistry, biotechnology and biomedical laboratories. Table of Contents......Page 7 Abbreviations......Page 14 1.1 Quantitative Determinations of Proteins......Page 16 1.1.1 LOWRY Protein Quantification......Page 17 1.1.2 BRADFORD Protein Determination......Page 21 1.1.3 Protein Determination in SDS-PAGE Sample Solutions......Page 22 1.1.4 Protein Determination Using Amido Black......Page 23 1.1.5 BCA Protein Determination......Page 24 1.1.6 KJELDAHL Protein Determination......Page 25 1.1.7 UV Photometric Assay of Protein Concentration......Page 26 1.2.1 SCHMIDT and THANNHAUSER DNA, RNA, and Protein Separation Procedure......Page 28 1.2.3 Diphenylamine DNA (Deoxyribose) Determination......Page 29 1.2.4 Quantitative DNA Determination with Fluorescent Dyes......Page 30 1.2.5 Determination of Nucleic Acids by UVAbsorption......Page 31 1.3.1 Determination of Inorganic Phosphate in Biologic Samples......Page 32 1.3.3 Phospholipid Determination......Page 33 1.4 Monosaccharide Determination......Page 34 1.5 Calculations in Quantitative Analysis......Page 35 2.1 Polyacrylamide Gel Electrophoresis Systems......Page 37 2.1.1 LAEMMLI SDS-Polyacrylamide Gel Electrophoresis......Page 40 2.1.2 SDS-Polyacrylamide Gel Electrophoresis at Neutral pH (NuPAGE)......Page 45 2.1.3 SDS-Polyacrylamide Gel Electrophoresis According to WEBER, PRINGLE, and OSBORN......Page 46 2.1.4 Urea-SDS-Polyacrylamide Gel Electrophoresis for the Separation of Low Molecular Weight Proteins......Page 48 2.1.5 TRICINE-SDS-Polyacrylamide Gel Electrophoresis for Proteins and Oligopeptides in the Range of 1000–50 000 Daltons......Page 49 2.1.6 SDS-Polyacrylamide Gel Electrophoresis at pH 2.4......Page 50 2.1.7 Urea-Polyacrylamide Gel Electrophoresis for Basic Proteins at pH 2......Page 51 2.1.8 Anodic Discontinuous Polyacrylamide Gel Electrophoresis (Native PAGE)......Page 52 2.1.9 Cathodic Discontinuous Polyacrylamide Gel Electrophoresis (Native PAGE)......Page 53 2.1.10 Affinity Electrophoresis......Page 54 2.1.11 Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE; IEF followed by SDS-PAGE)......Page 55 2.2.1 Non-denaturating Nucleic Acid Electrophoresis......Page 59 2.2.2 Denaturating Nucleic Acid Electrophoresis......Page 60 2.2.3 Identification of Phosphoamino Acids (Paper Electrophoresis)......Page 62 2.3.1 Marker Dyes for Monitoring Electrophoresis......Page 63 2.3.2 Marker Proteins for the Polyacrylamide Gel Electrophoresis......Page 64 2.3.3 Covalently Colored Marker Proteins......Page 66 2.4.1 Staining with Organic Dyes......Page 67 2.4.2 Silver Staining of Proteins in Gels......Page 70 2.4.3 Copper Staining of SDS-PAGE Gels......Page 75 2.4.4 Staining of Glycoproteins and Polysaccharides in Gels......Page 76 2.4.5 Staining of Blotted Proteins on Membranes......Page 77 2.5.1 Preparative Electroelution of Proteins from Polyacrylamide Gels......Page 80 2.5.2 Removal of SDS......Page 81 2.5.3 Electrotransfer of Proteins onto Membranes (Electroblotting; Western Blot): Semi-dry Blotting......Page 82 2.5.4 Immunochemical Detection of Antigens After Electrotransfer (Immunoblotting)......Page 84 2.5.5 Chemiluminescence Detection on Blotting Membranes......Page 88 2.5.6 Carbohydrate-Specific Glycoprotein Detection After Electrotransfer......Page 89 2.5.7 General Carbohydrate Detection on Western Blots......Page 90 2.5.8 Affinity Blotting......Page 91 2.5.9 Transfer of Nucleic Acids (SOUTHERN and Northern Blot)......Page 92 2.6 Drying of Electrophoresis Gels......Page 93 2.7 Autoradiography of Radioactive Labeled Compounds in Gels......Page 94 3.1.1 Identification of the N-terminal Amino Acid in Polypeptides (TLC of Modified Amino Acids)......Page 97 3.1.3 Gradient Thin-Layer Chromatography of Nucleotides......Page 99 3.1.4 Identification of Phosphates on TLC Plates......Page 101 3.1.5 Lipid Extraction and TLC of Lipids......Page 102 3.2 Hints for Column Chromatography of Proteins......Page 103 3.3 Gel Permeation Chromatography (GPC; Gel Filtration, GF; Size-Exclusion Chromatography, SEC)......Page 107 3.3.1 Selection of Supports......Page 110 3.3.3 Sample Application and Chromatographic Separation (Elution)......Page 111 3.3.4 Cleaning and Storage......Page 112 3.3.6 Removing of Unbound Biotin After Conjugation by Gel Filtration ("Desalting")......Page 113 3.4 Ion Exchange Chromatography (IEC)......Page 116 3.4.1 Preparation of Ion Exchange Supports......Page 117 3.4.3 Sample Application......Page 118 3.4.5 Cleaning and Regeneration......Page 119 3.4.6 High-Performance Ion Exchange Chromatography (HPIEC) of Mono- and Oligosaccharides......Page 120 3.5.1 Capacity Test......Page 121 3.5.4 Analytical HPLC of Hapten-Protein Conjugates......Page 122 3.6 Affinity Chromatography (AC)......Page 123 3.6.1 Cyanogen Bromide Activation of Polysaccharide-Based Supports......Page 127 3.6.2 Coupling to Cyanogen Bromide-Activated Gels......Page 128 3.6.4 Immobilization of Monosaccharides (Fucose)......Page 133 3.6.5 Activation with Divinylsulfone......Page 134 3.6.7 Covalent Coupling of Biotin (Biotin-Avidin/Streptavidin System)......Page 135 3.6.8 Metal Chelate Chromatography of Proteins Containing His[sub(6)]-Tag......Page 137 3.7.2 Salting Out......Page 138 3.7.3 Precipitation Using Organic Substances......Page 139 3.7.4 Lyophilization (Freeze Drying)......Page 140 3.7.5 Ultrafiltration......Page 141 4.1 Conjugation of Haptens (Peptides) to Carrier Proteins......Page 143 4.1.2 Conjugation of MCA-Gly Peptides to SH-Carrying Proteins......Page 146 4.1.3 Conjugation of Sulfhydryl Peptides Using 4-(N-Maleimidomethyl)-Cyclohexane-1-Carbonic Acid N-Hydroxysuccinimide Ester (SMCC)......Page 147 4.1.5 Carbodiimide Coupling of Peptides to Carrier Proteins with 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide (EDAC, EDC)......Page 148 4.1.6 Conjugation of Horseradish Peroxidase (Glycoproteins) by Periodate Oxidation......Page 149 4.1.7 Conjugation of Peptides to Carrier Proteins Using Glutaraldehyde (Two-Step Procedure)......Page 150 4.1.8 Conjugation of HRP to Antibodies with Glutaraldehyde......Page 151 4.1.10 Labeling of Immunoglobulins with Fluorescent Dyes......Page 152 4.1.11 Protein-Colloidal Gold Conjugates......Page 155 4.2 Immunization of Laboratory Animals......Page 157 4.3 Ammonium Sulfate Fractionation of Immunoglobulins......Page 158 4.4 Removal of Unspecific Immunoreactivities......Page 160 4.5 Preparation of Egg Yolk IgY Fraction......Page 162 4.6.1 F(ab ́)2 Fragments from IgG......Page 163 4.7 HEIDELBERGER Curve (Precipitin Curve)......Page 164 4.8.2 Preparation of Slides......Page 165 4.8.4 Visualization of the Precipitin Lines......Page 166 4.9 Immunoprecipitation of Antigens......Page 167 4.10 Immunoelectrophoresis......Page 168 4.11 Counterelectrophoresis......Page 169 4.12 Dot-Blot Assay......Page 170 4.13 Enzyme Immunosorbent Assay (EIA, ELISA)......Page 171 4.13.1 Indirect EIA with HRP Conjugate......Page 172 4.13.2 Determination of Enzyme Activity by ELISA......Page 173 4.13.3 Isotype Determination by EIA (AP Conjugate)......Page 174 5.1 Speed vs Centrifugal Force Graphs......Page 175 5.2 Differential Centrifugation......Page 178 5.3 Density Gradient Centrifugation......Page 179 5.3.1 Pre-formed Discontinuous Gradient Centrifugation: Isolation of Liver Cell Nuclei......Page 180 5.3.2 Sucrose Gradient Centrifugation: Preparation of Surface Membranes (Sarcolemma, SL) of Heart Muscle Cells......Page 181 5.3.3 RNA Separation by Non-Denaturating Sucrose Density Gradient Centrifugation......Page 189 5.3.4 Denaturating RNA Gradient Centrifugation......Page 190 5.3.5 Isopycnic Centrifugation......Page 191 6 Radioactive Labeling......Page 195 6.1 Radioactive Decay......Page 196 6.2 Decay Tables for 32-Phosphorus, 35-Sulfur, and 125-Iodine......Page 197 6.3 Enzymatic [[sup(32)]P]-Phosphate Incorporation into Proteins......Page 199 6.4.1 Chloramine-T Protocol......Page 201 6.5 Scintillation Cocktails for Liquid Scintillation Counting......Page 202 7.1 Theoretical Considerations......Page 204 7.2 Plot for Buffer Calculations......Page 211 7.4 Buffer Recipes......Page 212 8.1 Concentration Units......Page 221 8.2 Conversion Factors for SI Units......Page 222 8.3 Data of Frequently Used Substances......Page 224 8.4 Protein Data......Page 228 8.5 Protease Inhibitors......Page 233 8.6 Single-Letter Codes and Molecular Masses of Amino Acids......Page 234 8.8 Detergents ("Surfactants")......Page 237 8.9 Refractive Index and Density of Sucrose Solutions......Page 240 8.10 Ammonium Sulfate Saturation Table......Page 241 8.11 Diluted Solutions......Page 243 8.12 Mixture Rule......Page 244 9.1.1 Mean and Related Functions......Page 245 9.1.2 Correlation: Linear Regression......Page 246 9.1.3 The t-test (Student's Test)......Page 248 9.2.1 Receptor–Ligand Binding......Page 249 9.2.2 Enzyme Kinetics......Page 252 9.2.3 Determination of Molecular Mass by SDS-PAGE......Page 255 9.4 Software for the Lab......Page 256 9.4.3 Other Software.......Page 257 9.4.4 Selected Internet Links......Page 258 C......Page 259 H......Page 260 O......Page 261 S......Page 262 Z......Page 263 This Book Presents Proven Lab Procedures And Practical Hints For Research In Analytical And Preparative Biochemistry, And Offers Convenient Key Data In Numerous Tables. Coverage Includes Quantitative Methods; Electrophoresis; Chromatographic Protocols; Immunochemical Protocols; Centrifugation; And Radioactivity. In Additional Chapters, Tables Offer Quick Access To A Broad Array Of Useful Information, Including Si Units Conversion Factors; Detergent, Protein And Nucleotide Data; And The Basic Principles Of Statistics And Enzyme And Receptor Kinetics Are Reviewed. This First English-language Edition Of A Successful German-language Manual Is A Valuable Resource For Students And Working Professionals In Biochemistry, Biotechnology And Biomedical Laboratories. Martin Holtzhauer. Includes Bibliographical References And Index. Mode Of Access: World Wide Web.