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Detailed Contents

Laboratory Techniques in Biochemistry

All of our knowledge of biochemistry is the outcome of experiments. For the most part,
this text presents biochemical knowledge as established fact, but students should never lose
sight of the obligatory connection between scientific knowledge and its validation by obser-
vation and analysis. The path of discovery by experimental research is often indirect, tortu-
ous, and confounding before the truth is realized. Laboratory techniques lie at the heart of
scientific inquiry, and many techniques of biochemistry are presented within these pages to
foster a deeper understanding of the biochemical principles and concepts that they reveal.

Recombinant DNA Techniques Fluoro-substituted analogs as therapeutic agents 913

Restriction endonuclease digestion of DNA 329-331 “Knockout” mice 971
Restriction mapping 331-332
Nucleotide sequencing 337-341 Isolation/Purification of Macromolecules
Nucleic acid hybridization 354-355 Ion exchange chromatography 107-108
Chemical synthesis of oligonucleotides 364-365 Protein purification protocols 105-107
Cloning; recombinant DNA constructions 379-385 Dialysis and ultrafiltration 107
Construction of genomic DNA libraries 385-386 Size exclusion chromatography 108-109
Combinatorial libraries of synthetic oligomers 385 SDS-polyacrylamide gel electrophoresis 108-109
Screening DNA libraries by colony hybridization 386 Isoelectric focusing 110
mRNA isolation 387 Two-dimensional gel electrophoresis 110
Construction of cDNA libraries 387-391 Hydrophobic interaction chromatography 110
Expressed sequence tags 392 High-performance liquid chromatography 111
Southern blotting 388-389 Affinity chromatography 111
Gene chips (DNA microarrays) 392 Ultracentrifugation 111
Protein expression from cDNA inserts 392-394 Fractionation of cell extracts by centrifugation 111
Screening protein expression libraries with antibodies 394-395
Two-hybrid systems to identify protein:protein interactions 396-397 Analyzing the Physical and Chemical Properties  
Reporter gene constructs 396 of Biomolecules
Polymerase chain reaction (PCR) 397-398
Titration of weak acids 39-43
In vitro mutagenesis 399
Preparation of buffers 43-46
Measurement of standard reduction potentials 70-73
Probing the Function of Biomolecules Edman degradation 86-87, 111
Green fluorescent protein 88 Estimation of protein concentration 106
Plotting enzyme kinetic data 420-421 Amino acid analysis of proteins 111
Enzyme inhibition 423-429 Amino acid sequence determination 112-122
Optical trapping to measure molecular forces 526 Mass spectrometry of proteins 118-122
Isotopic tracers as molecular probes 566-567 Peptide mass fingerprinting 121-122
RNAi 399 Solid-phase peptide synthesis 130-132
NMR spectroscopy 567 Membrane lipid phase transitions 279-281
Transgenic animals 976 DNA nanotechnology 321
DNA footprinting 998 Nucleic acid hydrolysis 326-329
DNA sequencing 337-341
Techniques Relevant to Clinical Biochemistry Single-molecule DNA sequencing 340-341
Gene therapy 400-403 Density gradient (isopycnic) centrifugation 356
Metabolomic analysis 570-572
Fluxomics 571-572
Tumor diagnosis with positron emission tomography (PET) 600
Glucose monitoring devices 720

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