Inhalt

1 General Aspects of Nonradioactive Labeling and Detection.- 1.1 Introduction.- 1.2 The Concept of Nonradioactive Bioanalytics..- 1.3 Direct and Indirect Labeling and Detection Systems.- 1.3.1 Direct Systems.- 1.3.2 Indirect Systems.- 1.4 Labeling Methods.- 1.4.1 Enzymatic Labeling.- 1.4.2 Photochemical Labeling.- 1.4.3 Chemical Labeling.- 1.5 Detection Methods.- 1.5.1 Direct Detection Systems.- 1.5.2 Indirect Detection Systems.- 1.5.2.1 Optical Detection.- 1.5.2.2 Luminescence Detection.- 1.5.2.3 Fluorescence Detection.- 1.6 Guide of the Use of Information in this Book.- I Standard Nonradioactive Labeling and Detection Systems.- 2 Overview of Nonradioactive Labeling Systems.- 3 The Digoxigenin: Anti-Digoxigenin (DIG) System.- 3.1 Overview.- 3.2 Labeling and Detection of Nucleic Acids.- 3.2.1 Principle and Applications.- 3.2.2 Reaction Scheme.- 3.2.3 Random-Primed Labeling with DIG-[11]-dUTP and Klenow Enzyme.- 3.2.4 PCR-Guided Synthesis of Vector-Free, DIG-Labeled DNA Probe with DIG-[11]-dUTP.- 3.2.5 RNA Labeling by Run-off Transcription with DIG-[11]-UTP and SP6, T7 or T3 RNA Polymerase.- 3.2.6 Oligonucleotide Tailing with DIG-[11]-dUTP/dATP and Terminal Transferase.- 3.2.7 Oligonucleotide 3? End Labeling with DIG-[11]-ddUTP and Terminal Transferase.- 3.2.8 Photolabeling off DNA or RNA with PhotoDIG.- 3.2.9 Hybridization with DIG-Labeled DNA or DIG-Labeled Oligonucleotides.- 3.2.10 Hybridization with DIG-Labeled RNA.- 3.2.11 Optical Detection of DIG Modification with BCIP/NBT.- 3.2.12 Chemiluminescent Detection of DIG Modification with Lumigen TMPPD or Lumiphostm TM530.- 3.2.13 Special Hints for Application and Troubleshooting.- 3.3 Labeling and Detection of Proteins and Glycoproteins.- 3.3.1 Principle and Applications.- 3.3.2 Labeling and Detection of Proteins/Peptides.- 3.3.3 Labeling and Detection of Glycoconjugates.- 3.3.4 General Labeling and Detection of Proteins/Peptides with DIG-Ester and DIG-Maleimide.- 3.3.5 Selective Labeling of Sulfhydryl Groups.- 3.3.6 Selective Labeling of Disulfides.- 3.3.7 Special Hints for Application and Troubleshooting.- 3.3.8 General Labeling and Detection of Glycoconjugates with DIG-Hydrazide.- 3.3.9 Special Hints for Application and Troubleshooting.- 3.3.10 Sialic Acid-Specific Oxidation.- 3.3.11 Special Hints for Application and Troubleshooting.- 3.3.12 Selective Detection of Terminal Galactose Residues.- 3.3.13 Special Hints for Application and Troubleshooting.- 3.3.14 Labeling of Glycoconjugates with DIG-Lectins.- 3.3.15 Special Hints for Application and Troubleshooting.- 4 The Biotin System.- 4.1 Labeling and Detection of Nucleic Acids.- 4.1.1 Principle and Applications.- 4.1.1.1 Labeling of Nucleic Acids with Biotin.- 4.1.1.2 Biotinylated Nucleotides.- 4.1.2 Methods of Labeling DNA.- 4.1.2.1 Biotin Labeling of DNA by Nick Translation.- 4.1.2.2 Random Primer Labeling of DNA with Biotin.- 4.1.2.3 Polymerase Chain Reaction.- 4.1.2.4 Labeling with Photobiotin.- 4.1.3 Labeling of Synthetic Oligonucleotides.- 4.1.3.1 Enzymatic Labeling.- 4.1.3.2 Chemical Synthesis of Biotinylated Oligonucleotides.- 4.1.4 Methods of Labeling RNA.- 4.1.5 Hybridization and Detection of Biotinylated Probes.- 4.1.5.1 Hybridization.- 4.1.5.2 Hybridization of Synthetic Oligonucleotide Probes.- 4.1.5.3 Binding the Streptavidin-Alkaline Phosphatase Conjugate.- 4.1.5.4 Chromogenic Detection of Biotinylated Probes with Alkaline Phosphatase.- 4.1.5.5 Chemiluminescent Detection of Biotinylated Probes with Alkaline Phosphatase.- 4.1.6 Summary.- 4.2 Labeling and Detection of Proteins and Glycoproteins.- 4.2.1 Principle and Applications.- 4.2.2 Biotinylation of Proteins via Lysines.- 4.2.3 Special Hints for Application and Troubleshooting.- 4.2.4 Biotinylation of Proteins via Cysteines.- 4.2.5 Special Hints for Application and Troubleshooting.- 4.2.6 Biotinylation of Proteins via Tyrosines or Histidines.- 4.2.7 Special Hints for Application and Troubleshooting.- 4.2.8 Biotinylation of Proteins via Aspartatic and Glutamic Acids.- 4.2.9 Special Hints for Application and Troubleshooting.- 4.2.10 Biotinylation of Glycoproteins via Sugar Residues.- 4.2.11 Special Hints for Application and Troubleshooting.- 5 In Vivo Labeling of DNA Probes with 5-BrdU.- 5.1 Principle and Applications.- 5.2 Reaction Scheme.- 5.3 In Vivo Recombinant M13 DNA Labeling with 5-BrdU, Hybridization, and Immunoenzymatic Detection.- 5.4 Special Hints for Application and Troubleshooting.- 6 The Sulfone System.- 6.1 Principle and Applications.- 6.2 The Photo-ChemiProbe Procedure: Labeling by Sulfonation, Hybridization, and Chemiluminescence Visualization.- 6.2.1 BCIP/NBT as an Alternative Chromogenic System.- 6.3 Special Hints for Application and Troubleshooting.- 6.4 Labeling of Oligonucleotide Primers for Use in PCR and the Use of Amplified Products as a Labeled Probe.- 7 Colloidal Gold as a Marker in Molecular Biology: The Use of Ultra-Small Gold Probes.- 7.1 Principle and Applications.- 7.1.1 Limitations of the Gold Marker System.- 7.1.2 Improvement by Reduction of Particle Size.- 7.2 Reaction Scheme.- 7.3 Indirect Immunogold Silver Staining.- 7.4 Special Hints for Application and Troubleshooting.- 7.5 Selected Example: Immunodetection of Gonadatropic Hormone in Catfish Pituitary.- 8 Direct Peroxidase Labeling of Hybridization Probes and Chemiluminescence Detection.- 8.1 Principle and Applications.- 8.2 Reaction Scheme.- 8.3 Directly Labeled Nucleic Acid (Long) Probes.- 8.4 Oligonucleotide Probes.- 8.5 Special Hints for Application and Troubleshooting.- 9 A Highly Sensitive Method for Detecting Peroxidase in In Situ Hybridization or Immunohistochemical Assays.- 9.1 Principle and Applications.- 9.2 Reaction Scheme.- 9.3 In Situ Detection of Peroxidase.- 9.4 Special Hints for Application and Troubleshooting.- 10 The SNAP System.- 10.1 Principle and Applications.- 10.2 Colorimetric Detection of Mycobacteria on Filters.- 10.3 Colorimetric Detection of Human Papillomavirus In Situ.- 10.4 Chemiluminescent Detection of Human DNA Fingerprints.- 10.5 Fluorescence Detection of Human Immunodeficiency Virus Using Sandwich Hybridization.- II Specialized Nonradioactive Detection Systems.- 11 Overview of Colorimetric, Chemiluminometric, and Fluorimetric Detection Systems.- 11.1 Principle and Applications.- 11.2 Substrates for Hydrolases.- 11.2.1 Direct Substrates.- 11.2.2 Indirect Substrates.- 11.3 Substrates for Peroxidase.- 11.4 Applications of Direct Labels.- 12 Colorimetric Systems.- 12.1 Indigo/Tetrazolium Dyes.- 12.1.1 Principle and Applications.- 12.1.2 Detection with BCIP/NBT.- 12.2 Azo Dyes.- 12.2.1 Principle and Applications.- 12.2.2 Detection with Azo Dyes.- 12.2.3 Special Hints for Application and Troubleshooting.- 13 Luminescence Systems.- 13.1 Chemiluminescence: Luminol.- 13.1.1 Principle and Applications.- 13.1.2 Chemiluminescent Labeling and Detection with Horseradish Peroxidase and Luminol.- 13.1.3 Special Hints for Application and Troubleshooting.- 13.2 Chemiluminescence: Properties of 1,2-Dioxetane Chemiluminescence.- 13.3 Electrochemiluminescence: Ruthenium Complexes.- 13.3.1 Principle and Applications.- 13.3.2 Detection of PCR Products Using an Electrochemiluminescence Assay.- 13.3.3 Special Hints for Application and Troubleshooting.- 13.4 Bioluminescence: Luciferin.- 13.4.1 Principle and Applications.- 13.4.2 Reaction Scheme.- 13.4.3 Bioluminescence-Enhanced Detection.- 13.4.3.1 Enzymatic Labeling with Alkaline Phosphatase..- 13.4.3.2 Enzymatic Labeling with ?-Galactosidase.- 13.4.4 Special Hints for Application and Troubleshooting.- 14 Fluorescence Systems.- 14.1 Labeling of Biomolecules with Fluorescein, Resorufin, and Rhodamine.- 14.1.1 Principle and Applications.- 14.1.2 Fluorescent Labeling of Proteins.- 14.1.3 Special Hints for Application and Troubleshooting.- 14.2 Time-Resolved Fluorescence.- 14.2.1 Principle and Applications.- 14.2.2 Reaction Scheme.- 14.2.3 DNA Labeling with Biotin, Hybridization, and Detection.- 14.2.4 Special Hints for Ap…