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By: Martin Holtzhauer

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Paperback | 19 July 2006

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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.

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Non-Fiction Engineering & Technology Industrial Chemistry & Manufacturing Technologies Industrial Chemistry Chemical Engineering Science Biology, Life Sciences Biochemistry Medicine Medicine in General Medical Equipment & Techniques Medical Laboratory Testing & Techniques

Chemistry Analytical Chemistry Biochemical Engineering Biotechnology Medical Research

Abbreviations	p. XVII
Quantitative Methods	p. 1
Quantitative Determinations of Proteins	p. 1
Lowry Protein Quantification	p. 2
Standard Procedure	p. 2
Modification	p. 3
Micromethod on Microtest Plates	p. 4
Protein Determination in the Presence of Interfering Substances	p. 5
Bradford Protein Determination	p. 6
Protein Determination in SDS-PAGE Sample Solutions	p. 7
Protein Determination Using Amido Black	p. 8
BCA Protein Determination	p. 9
BCA Standard Procedure	p. 9
BCA Micromethod	p. 9
Kjeldahl Protein Determination	p. 10
UV Photometric Assay of Protein Concentration	p. 11
Quantitative Determination of Nucleic Acids	p. 13
Schmidt and Thannhauser DNA, RNA, and Protein Separation Procedure	p. 13
Orcin RNA (Ribose) Determination	p. 14
Diphenylamine DNA (Deoxyribose) Determination	p. 14
Quantitative DNA Determination with Fluorescent Dyes	p. 15
Determination of Nucleic Acids	p. 16
Quantitative Phosphate Determinations	p. 17
Determination of Inorganic Phosphate in Biologic Samples	p. 17
Determination of Total Phosphate	p. 18
Phospholipid Determination	p. 18
Monosaccharide Determination	p. 19
Calculations in Quantitative Analysis	p. 20
Electrophoresis	p. 23
Polyacrylamide Gel Electrophoresis Systems	p. 23
Laemmli SDS-Polyacrylamide Gel Electrophoresis	p. 26
SDS-Polyacrylamide Gel Electrophoresis at Neutral pH (NuPAGE)	p. 31
SDS-Polyacrylamide Gel Electrophoresis According to Weber, Pringle, and Osborn	p. 32
Urea-SDS-Polyacrylamide Gel Electrophoresis for the Separation of Low Molecular Weight Proteins	p. 34
TRICINE-SDS-Polyacrylamide Gel Electrophoresis for Proteins and Oligopeptides in the Range of 1000-50 000 Daltons	p. 35
SDS-Polyacrylamide Gel Electrophoresis at pH 2.4	p. 36
Urea-Polyacrylamide Gel Electrophoresis for Basic Proteins at pH 2	p. 37
Anodic Discontinuous Polyacrylamide Gel Electrophoresis (Native PAGE)	p. 38
Cathodic Discontinuous Polyacrylamide Gel Electrophoresis ((Native PAGE)	p. 39
Affinity Electrophoresis	p. 40
Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE; IEF followed by SDS-PAGE)	p. 41
First Dimension: Isoelectric Focusing (IEF)	p. 42
Second Dimension: SDS-PAGE (Acrylamide Gradient Gel)	p. 44
Agarose and Paper Electrophoresis	p. 45
Non-denaturating Nucleic Acid Electrophoresis	p. 45
Denaturating Nucleic Acid Electrophoresis	p. 46
Identification of Phosphoamino Acids (Paper Electrophoresis)	p. 48
Aid in Electrophoresis	p. 49
Marker Dyes for Monitoring Electrophoresis	p. 49
Anodic Systems	p. 49
Cathodic Systems	p. 49
Marker Proteins for the Polyacrylamide Gel Electrophoresis	p. 50
Covalently Colored Marker Proteins	p. 52
Staining Protocols	p. 53
Staining with Organic Dyes	p. 53
Amido Black 10 B	p. 54
Coomassie Brilliant Blue R250 and G250	p. 54
Coomassie Brilliant Blue R250 Combined with Bismarck Brown R	p. 55
Fast Green FCF	p. 55
Stains All	p. 56
Staining of Proteolipids, Lipids, and Lipoproteins	p. 56
Silver Staining of Proteins in Gels	p. 56
Citrate/Formaldehyde Development	p. 57
Alkaline Development	p. 58
Silver Staining Using Tungstosilicic Acid	p. 58
Silver Staining of Proteins: Formaldehyde Fixation	p. 59
Silver Staining of Glycoproteins and Polysaccharides	p. 60
Enhancement of Silver Staining	p. 60
Reducing of Silver-Stained Gels	p. 61
Copper Staining of SDS-PAGE Gels	p. 61
Staining of Glycoproteins and Polysaccharides in Gels	p. 62
Staining with Schiff's Reagent (PAS Staining)	p. 62
Staining with Thymol	p. 63
Staining of Blotted Proteins on Membranes	p. 63
Staining on Nitrocellulose with Dyes	p. 63
Staining on Nitrocellulose with Colloidal Gold	p. 64
Staining on PVDF Blotting Membranes with Dyes	p. 65
Electroelution from Gels	p. 66
Preparative Electroelution of Proteins from Polyacrylamide Gels	p. 66
Removal of SDS	p. 67
Electrotransfer of Proteins onto Membranes (Electroblotting; Western Blot): Semi-dry Blotting	p. 68
Immunochemical Detection of Antigens After Electrotransfer (Immunoblotting)	p. 70
Detection Using Horseradish Peroxidase (HRP)	p. 72
Detection Using Alkaline Phosphatase (AP)	p. 73
Chemiluminescence Detection on Blotting Membranes	p. 74
Chemiluminescence Using HRP	p. 74
Chemiluminescence Using AP	p. 74
Carbohydrate-Specific Glycoprotein Detection After Electrotransfer	p. 75
General Carbohydrate Detection on Western Blots	p. 76
Affinity Blotting	p. 77
Transfer of Nucleic Acids (Southern and Northern Blot)	p. 78
Drying of Electrophoresis Gels	p. 79
Autoradiography of Radioactive Labeled Compounds in Gels	p. 80
Chromatography	p. 83
Thin-Layer Chromatography	p. 83
Identification of the N-terminal Amino Acid in Polypeptides (TLC of Modified Amino Acids	p. 83
Thin-Layer Chromatography of Nucleoside Phosphates	p. 85
Gradient Thin-Layer Chromatography of Nucleotides	p. 85
Identification of Phosphates on TLC Plates	p. 87
Lipid Extraction and TLC of Lipids	p. 88
Hints for Column Chromatography of Proteins	p. 89
Gel Permeation Chromatography (GPC; Gel Filtration, GF; Size-Exclusion Chromatography, SEC)	p. 93
Selection of Supports	p. 96
Filling of a Gel Filtration Column	p. 97
Sample Application and Chromatographic Separation (Elution)	p. 97
Cleaning and Storage	p. 98
Determination of Void Volume V[subscript 0] and Total Volume V[subscript t]	p. 99
Removing of Unbound Biotin After Conjugation by Gel Filtration ("Desalting")	p. 99
Ion Exchange Chromatography (IEC)	p. 102
Preparation of Ion Exchange Supports	p. 103
Capacity Test	p. 104
Sample Application	p. 104
Elution	p. 105
Cleaning and Regeneration	p. 105
High-Performance Ion Exchange Chromatography (HPIEC) of Mono- and Oligosaccharides	p. 106
Hydrophobic Interaction Chromatography (HIC)	p. 107
Capacity Test	p. 107
Elution	p. 108
Regeneration	p. 108
Analytical HPLC of Hapten-Protein Conjugates	p. 108
Affinity Chromatography (AC)	p. 109
Cyanogen Bromide Activation of Polysaccharide-Based Supports	p. 113
Determination of the Degree of Activation	p. 114
Coupling to Cyanogen Bromide-Activated Gels	p. 114
Quantitative Determination of Coupled Diamine Spacers with 2,4,6-Trinitrobenzene Sulfonic Acid	p. 115
Quantitative Determination of Immobilized Protein	p. 116
Immobilization of Wheat Germ Agglutinin	p. 116
Affinity Purification of HRP	p. 117
Affinity Chromatography of Immunoglobulins on Immobilized Antibodies (Immunoaffinity Chromatography, IAC)	p. 117
Affinity Chromatography of Rabbit IgG on Protein-A Supports	p. 118
Activation of Sepharose with Epichlorohydrin	p. 119
Determination of Epoxy Residues	p. 119
Immobilization of Monosaccharides (Fucose)	p. 119
Activation with Divinylsulfone	p. 120
Coupling of Reactive Dyes to Polysaccharides (Dye-Ligand Chromatography)	p. 121
Covalent Coupling of Biotin (Biotin-Avidin/Streptavidin System)	p. 121
Metal Chelate Chromatography of Proteins Containing His[subscript 6]-Tag	p. 123
Concentration of Diluted Protein Solutions	p. 124
Acidic Precipitation	p. 124
Salting Out	p. 124
Precipitation Using Organic Substances	p. 125
Lyophilization (Freeze Drying)	p. 126
Ultrafiltration	p. 127
Immunochemical Protocols	p. 129
Conjugation of Haptens (Peptides) to Carrier Proteins	p. 129
Activation of Proteins with Traut's Reagent Yielding Proteins with Additional Free SH Groups	p. 132
Conjugation of MCA-Gly Peptides to SH-Carrying Proteins	p. 132
Conjugation of Sulfhydryl Peptides Using 4-(N-Maleimidomethyl)-Cyclohexane-l-Carbonic Acid N-Hydroxysuccinimide Ester (SMCC)	p. 133
[beta]-Galactosidase-Immunoglobulin Conjugate (Coupling via SH Groups)	p. 134
Enzyme Reaction of[beta]-Galactosidase	p. 134
Carbodiimide Coupling of Peptides to Carrier Proteins with 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide (EDAC, EDC)	p. 134
Conjugation of Horseradish Peroxidase (Glycoproteins) by Periodate Oxidation	p. 135
Conjugation of Peptides to Carrier Proteins Using Glutaraldehyde (Two-Step Procedure)	p. 136
Conjugation of HRP to Antibodies with Glutaraldehyde	p. 137
Alkaline Phosphatase-Immunoglobulin Conjugate (Glutaraldehyde Protocol)	p. 138
Enzymatic Reaction of Alkaline Phosphatase from Calf Intestine	p. 138
Labeling of Immunoglobulins with Fluorescent Dyes	p. 138
Protein-Colloidal Gold Conjugates	p. 141
Preparation of Colloidal Gold Sol	p. 141
Adsorption of Protein to Colloidal Gold	p. 142
Immunization of Laboratory Animals	p. 143
Ammonium Sulfate Fractionation of Immunoglobulins	p. 144
Removal of Unspecific Immunoreactivities	p. 146
Preparation of Tissue Powder (Liver Powder)	p. 148
Preparation of Egg Yolk IgY Fraction	p. 148
Antibody Fragmentation	p. 149
F(ab[prime])2 Fragments from IgG	p. 149
Fab[prime] Fragments (Rabbit)	p. 150
Fab Fragments (Rabbit)	p. 150
Heidelberger Curve (Precipitin Curve)	p. 150
Ouchterlony Double-Radial Immunodiffusion	p. 151
Purification of Agar	p. 151
Preparation of Slides	p. 151
Immunodiffusion	p. 152
Visualization of the Precipitin Lines	p. 152
Immunoprecipitation of Antigens	p. 153
Immunoelectrophoresis	p. 154
Counterelectrophoresis	p. 155
Dot-Blot Assay	p. 156
Enzyme Immunosorbent Assay (EIA, ELISA)	p. 157
Indirect EIA with HRP Conjugate	p. 158
Determination of Enzyme Activity	p. 159
Isotype Determination	p. 160
Centrifugation	p. 161
Speed vs Centrifugal Force Graphs	p. 161
Differential Centrifugation	p. 164
Density Gradient Centrifugation	p. 165
Pre-formed Discontinuous Gradient Centrifugation: Isolation of Liver Cell Nuclei	p. 166
Sucrose Gradient Centrifugation: Preparation of Surface Membranes (Sarcolemma, SL) of Heart Muscle Cells	p. 167
Determination of a Marker Enzyme: Ouabain-Sensitive Na,K-ATPase	p. 172
Receptor Determination: DHP Binding Sites on Surface Membranes	p. 173
Determination of the Dissociation and Association Kinetics of the DHP Receptors	p. 174
RNA Separation by Non-Denaturating Sucrose Density Gradient Centrifugation	p. 175
Denaturating RNA Gradient Centrifugation	p. 176
Isopycnic Centrifugation	p. 177
Purification of High Molecular Weight DNA in CsCl Gradients	p. 177
Cell Fractionation Using Percoll	p. 178
Preparation of Human Lymphocytes	p. 179
Radioactive Labeling	p. 181
Radioactive Decay	p. 182
Decay Tables for 32-Phosphorus, 35-Sulfur, and 125-Iodine	p. 183
Enzymatic [[superscript 32]P]-Phosphate Incorporation into Proteins	p. 185
Iodination with [[superscript 125]I]-Iodine Reagents	p. 187
Chloramine-T Protocol	p. 187
Iodination with Bolton-Hunter Reagent	p. 188
Scintillation Cocktails for Liquid Scintillation Counting	p. 188
Buffers	p. 191
Theoretical Considerations	p. 191
Plot for Buffer Calculations	p. 198
pH Indicators	p. 199
Buffer Recipes	p. 199
Commonly Used Buffers	p. 201
Buffers and Media for Tissue and Cell Culture and Organ Perfusion	p. 204
pH Calibration Buffers	p. 206
Volatile Buffers	p. 207
Tables	p. 209
Concentration Units	p. 209
Conversion Factors for SI Units	p. 210
Data of Frequently Used Substances	p. 212
Protein Data	p. 216
Protease Inhibitors	p. 221
Single-Letter Codes and Molecular Masses of Amino Acids	p. 222
Spectroscopic Data of Nucleotides	p. 225
Detergents ("Surfactants")	p. 225
Refractive Index and Density of Sucrose Solutions	p. 228
Ammonium Sulfate Saturation Table	p. 229
Diluted Solutions	p. 231
Mixture Rule	p. 232
Statistics and Data Analysis	p. 233
Statistical Equations	p. 233
Mean and Related Functions	p. 233
Correlation: Linear Regression	p. 234
The t-test (Student's Test)	p. 236
Data Analysis	p. 237
Receptor-Ligand Binding	p. 237
Enzyme Kinetics	p. 240
Determination of Molecular Mass by SDS-PAGE	p. 243
Diagnostic Sensitivity and Specificity	p. 244
Software for the Lab	p. 244
Data Analysis and Presentation	p. 245
Software for Statistics	p. 245
Other Software	p. 245
Selected Internet Links	p. 246
Subject Index	p. 247
Table of Contents provided by Ingram. All Rights Reserved.

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