Hardcover: 360 pages ; Dimensions (in inches): 0.90 x 9.80 x 6.88
Publisher: John Wiley & Sons; Book and CD-ROM edition (November 2002)

Preface  V 
1  Biochemical literature  1 
1.1  Accessing the biochemical literature  1 
1.1.1  Textbooks of biochemistry  2 
1.1.2  Current reviews of the biochemical literature  3 
1.1.3  The primary biochemical literature: scientific journals  4 
1.2  Access to the methodologically-based biochemical literature  6 
1.2.1  Monographs and series  6 
1.2.2  Methods-based biochemical journals  6 
1.3  Reference works and handbooks  7 
1.3.1  Reference works  7 
1.3.2  Handbooks and collected tables  8 
1.4  Literature searches  9 
1.4.1  Retrospective literature searches  9 
1.4.2  Current literature searches  9 
1.4.3  The Internet as an information resource  10 
1.5  Documentation of practical work  10 
1.5.1  The laboratory book  10 
1.5.2  The layout of the laboratory book  11 
1.6  Literature  11 
2  General laboratory procedures  13 
2.1  The biochemistry laboratory  13 
2.1.1  Apparatus needed in every biochemistry laboratory  13 
2.1.2  Apparatus that can be shared between several laboratories  14 
2.1.3  Miscellaneous small items  15 
2.1.4  Containers: glass, ceramic, metal and plastic in various sizes  15 
2.1.5  Disposables  15 
2.1.6  Safety equipment  16 
2.1.7  Standard reagents  16 
2.2  Routine biochemical procedures  17 
2.2.1  Safety requirements  17 
2.2.2  Cleaning of glass and plastic containers  19 
2.2.3  Weighing out solids  20 
2.2.4  Pipetting and measuring liquid volumes  20 
2.2.5  Preparation and storage of solutions: water quality and the purity of chemical reagents  23 
2.2.6  Thermostatting  24 
2.2.7  Shaking and stirring  25 
2.2.8  Use of pumps  26 
2.2.9  Buffers  27 
2.2.10  Supplementary reagents (preservatives, chelating agents, SH reagents and detergents)  31 
2.2.11  pH determination  32 
2.2.12  Conductivity measurements  33 
2.3  Working with radioactivity  33 
2.3.1  Radioactive isotopes and their decay  34 
2.3.2  Measurement of radioactivity  36 
2.3.3  Alternatives to radioactivity  43 
2.4  Literature  46 
3  Sample preparation  49 
3.1  Cell and tissue disruption  49 
3.1.1  General aspects of protein and nucleic acid isolation  50 
3.1.2  Mechanical homogenisation  52 
3.1.3  Non-mechanical homogenisation procedures  54 
3.2  Solubilisation  55 
3.3  Precipitation procedures for proteins and nucleic acids  56 
3.3.1  Precipitation of proteins  56 
3.3.2  Precipitation of nucleic acids  60 
3.4  Dialysis, ultrafiltration and lyophilisation  61 
3.4.1  Dialysis  61 
3.4.2  Ultrafiltration  63 
3.4.3  Lyophilisation  66 
3.5  Literature  67 
4  Separation methods  69 
4.1  Chromatography  69 
4.1.1  General principles and definitions  70 
4.1.2  Column chromatography  70 
4.1.3  Paper and thin layer chromatography  102 
4.1.4  Gas-liquid chromatography  104 
4.2  Electrophoresis  106 
4.2.1  General principles and definitions  106 
4.2.2  Cellulose acetate electrophoresis  109 
4.2.3  Gel electrophoresis  110 
4.2.4  Isoelectric focusing  124 
4.2.5  2D electrophoresis  127 
4.2.6  Blotting methods  129 
4.2.7  Evaluation and documentation of electrophoresis results  131 
4.2.8  Capillary electrophoresis  131 
4.3  Centrifugation  137 
4.3.1  Basic principles  138 
4.3.2  Centrifuges and rotors  140 
4.3.3  Analytical ultracentrifugation  145 
4.3.4  Preparative centrifugation  149 
4.4  Literature  151 
5  Analytical methods  155 
5.1  Protein analysis  155 
5.1.1  Determination of protein molar masses  155 
5.1.2  Quantitation of proteins  157 
5.1.3  Amino acid analysis  162 
5.1.4  End group determination  163 
5.1.5  Edman degradation  164 
5.1.6  Peptide mapping  166 
5.1.7  Co- and post-translational modification  171 
5.1.8  Chemical modification of proteins  173 
5.1.9  Structural analysis of proteins  182 
5.1.10  Protein stability  184 
5.1.11  Peptide synthesis and in vitro protein synthesis  187 
5.2  Nucleic acid analysis  190 
5.2.1  Determination of nucleic acid concentration  190 
5.2.2  Determination of nucleic acid size  191 
5.2.3  Base composition  191 
5.2.4  Restriction mapping  191 
5.2.5  Detection of specific DNA and RNA sequences by Southern and Northern blotting  193 
5.2.6  Detection of specific DNA and RNA sequences by the polymerase chain reaction (PCR)  194 
5.2.7  Nucleic acid sequencing  199 
5.2.8  Determination of the stability of double stranded nucleic acids  202 
5.2.9  Oligonucleotide synthesis  204 
5.2.10  Labelling and chemical modification of nucleic acids  207 
5.3  Enzymatic analysis  208 
5.3.1  Direct determination of metabolite concentrations  208 
5.3.2  Determination of metabolite concentrations by coupled measurements  209 
5.3.3  Determination of enzyme activity  210 
5.4  Literature  212 
6  Immunological methods  219 
6.1  Antibodies  219 
6.1.1  Antibody structure  220 
6.1.2  Antibody production  221 
6.1.3  Antibody purification  224 
6.2  Antibody-antigen interactions  225 
6.2.1  Antibody-antigen interactions in solution  226 
6.2.2  Antibody-antigen interactions in gels  226 
6.2.3  Radioimmunoassay  230 
6.2.4  Enzyme-linked immunosorbent assay  230 
6.2.5  Western blotting and dot blotting  234 
6.2.6  Immunofluorescence and immunogold electron microscopy  235 
6.2.7  Fluorescence activated cell sorting  235 
6.3  Literature  236 
7  Biophysical methods  239 
7.1  Spectroscopy  239 
7.1.1  Absorption of light  240 
7.1.2  Spectrophotometers  243 
7.1.3  Fluorescence  248 
7.1.4  Vibrational spectroscopy  255 
7.1.5  Anisotropic spectroscopy  256 
7.1.6  Nuclear magnetic resonance spectroscopy  259 
7.1.7  Mass spectrometry  264 
7.2  Scattering techniques  267 
7.2.1  Light-scattering in solution  267 
7.2.2  Scattering with other radiation  273 
7.3  Interactions  275 
7.3.1  Equilibrium dialysis  275 
7.3.2  Binding studies using filtration methods  276 
7.3.3  Binding studies using chromatography, electrophoresis and centrifugation  277 
7.3.4  Biomolecular interaction analysis  279 
7.3.5  Binding studies using protection and interference  280 
7.3.6  Calorimetry  281 
7.3.7  Kinetics  283 
7.4  Determination of structure  286 
7.4.1  X-ray structural analysis  286 
7.4.2  Structural databases  291 
7.5  Literature  292 
8  Mathematical Methods  295 
8.1  Statistics  295 
8.1.1  Observations and variables  296 
8.1.2  Errors and mean values  297 
8.1.3  Distributions  299 
8.2  Quantitative evaluation of experimental results  305 
8.2.1  Analysis of binding  306 
8.2.2  Enzyme kinetics  307 
8.3  Sequence analysis  313 
8.3.1  Databases  314 
8.3.3  Database searching  315 
8.4  Literature  320 
9  Quantitative Analysis of Biochemical Data  321 
9.1  Introduction  321 
9.1.1  General principles of quantitative data analysis  321 
9.1.2  Experimental systems  322 
9.1.3  Measurement and signals  323 
9.1.4  Models  323 
9.1.5  Selection of appropriate models  324 
9.1.6  Parameters  325 
9.1.7  Essential steps in the analysis  325 
9.1.8  Fitting data by the method of least squares  326 
9.1.9  Global fitting of multiple data sets  329 
9.1.10  Introduction to error estimation  329 
9.1.11  Introduction to numerical integration  331 
9.2  Applications  332 
9.2.1  Linear regression  332 
9.2.2  Michaelis-Menten kinetics  332 
9.2.3  Dissociation kinetics  334 
9.2.4  Binding data  335 
9.2.5  Independent identical binding sites  336 
9.2.6  Analysis of simple binding data  337 
9.2.7  Independent non-identical binding sites  337 
9.2.8  Cooperative binding  338 
9.2.9  Association kinetics  339 
9.2.10  Pre-steady state kinetics  340 
9.2.11  pH dependence of enzyme catalysed reactions  341 
9.2.12  Analysis of competition experiments  343 
9.3  Guide to the compact disc  344 
   Appendix I: SI-Units  345 
   Appendix II: Conversions into SI-Units  346 
   Index  347