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2.1 Introduction ...32 2.2 Technical Procedures ...32 Chemical Synthesis ...35 2.3 Results and Discussion...38 ...Chemical Synthesis ...38 Binding to anti-CCK antisera and CCK-B receptors ...40 Binding to streptavidin agarose and photoelution ...40 Affinity chromatography ...41 2.4 Troubleshooting...43 References ...44 ...Purification of the Receptor for Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) using Biotinylated Ligands ...45 Summary ...45 3.1 Introduction ...46 ...3.2 Technical Procedures ...48 Preparation of a Biotinylated Ligand ...48 Preparation of Membrane Fraction ...50 Solubilization of the PACAP Receptor ...51 Partial Purification of the PACAP Receptor ...52 Affinity Purification of the PACAP Receptor ...54 Final Purification of the PACAP Receptor ...56 Receptor-Binding Assay ...57 3.3 Results and Discussion...58 ...3.4 Troubleshooting ...61 Impurity in PACAP27-Cys-NH...61 2 Low specific activity of the PACAP receptor despite a single band ...62 Acknowledgments ...62 References ...63 ...Photoreactive Biotinylated Peptide Ligands for Affinity Labeling ..." 65 Summary ...65 4.1 Introduction...66 . 4.2 Technical Procedures ..."
67 Synthesis of a trifunctional photoactivatable biotinylating reagent ...67 Synthesis of photo reactive biotinylated peptide hormones ...67 Site-specific incorporation of biotin and photo labels in separate steps ...69 Photoaffinity labeling ...72 4.3 Results and Discussion ...76 Synthesis of photoactivatable insulins with permanent biotin labels ...76 Applications: Insulin ...78 VI Examples for other applications ...78 4.4 Troubleshooting...79 References ...81 ...
Contents
1 An Introduction to Avidin-Biotin Technology and Options for Biotinylation.- 1.1 Introduction.- 1.2 Technical Procedures.- 1.3 Troubleshooting.- Acknowledgments.- References.- 2 Synthesis of Photocleavable Biotinylated Ligands and Application for Affinity Chromatography.- Summary.- 2.1 Introduction.- 2.2 Technical Procedures.- 2.3 Results and Discussion.- 2.4 Troubleshooting.- References.- 3 Purification of the Receptor for Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) using Biotinylated Ligands.- Summary.- 3.1 Introduction.- 3.2 Technical Procedures.- 3.3 Results and Discussion.- 3.4 Troubleshooting.- Acknowledgments.- References.- 4 Photoreactive Biotinylated Peptide Ligands for Affinity Labeling.- Summary.- 4.1 Introduction.- 4.2 Technical Procedures.- 4.3 Results and Discussion.- 4.4 Troubleshooting.- References.- 5 Immunoprecipitation of Biotinylated Cell Surface Proteins.- Summary.- 5.1 Introduction.- 5.2 Technical Procedures.- 5.3 Results and Discussion.- 5.4 Troubleshooting.- Acknowledgments.- References.- 6 Biotinylation and Chemical Cross-Linking of Membrane Associated Molecules.- Summary.- 6.1 Introduction.- 6.2 Technical Procedures.- 6.3 Results and Discussion.- 6.4 Troubleshooting.- Acknowledgments.- References.- 7 Preparation of Biotinylated Lectins and Application in Microtiter Plate Assays and Western Blotting.- Summary.- 7.1 Introduction.- 7.2 Technical Procedures.- 7.3 Results and Discussion.- 7.4 Troubleshooting.- Acknowledgements.- References.- 8 Biotin-Labeling of Poly(ADP-ribose) in Poly(ADP-ribose)-Protein Interactions.- Summary.- 8.1 Introduction.- 8.2 Technical Procedures.- 8.3 Results and Discussion.- 8.4 Troubleshooting.- Acknowledgments.- References.- 9 Preparation and Use of Biotinylated Probes for the Detection and Characterisation of Serine Proteinase and Serine Proteinas Inhibitory Proteins.- Summary.- 9.1 Introduction.- 9.2 Technical Procedures.- 9.3 Results and Discussion.- 9.4 Troubleshooting.- Acknowledgments.- References.- 10 Avidin/Biotin-Mediated Conjugation of Antibodies to Erythrocytes: An Approach for Immunoerythrocyte Exploration in vivo.- Summary.- 10.1 Introduction.- 10.2 Technical Procedures.- 10.3 Results and Discussion.- 10.4 Troubleshooting.- Acknowledgments.- References.- 11 Biotin in vitro Translation: A Nonradioactive Method for the Synthesis of Biotin-Labeled Proteins in a Cell-Free System.- Summary.- 11.1 Introduction.- 11.2 Technical Procedures.- 11.3 Results and Discussion.- 11.4 Troubleshooting.- Acknowledgments.- References.- 12 Nonradioactive Detection of Nucleic Acids with Biotinylated Probes.- Summary.- 12.1 Introduction.- 12.2 Technical procedures.- 12.3 Results and discussion.- 12.4 Troubleshooting.- References.- 13 Biotin-Labeled Riboprobes to Study RNA-Binding Proteins.- Summary.- 13.1 Introduction.- 13.2 Technical Procedures.- 13.3 Results and Discussion.- 13.4 Troubleshooting.- Acknowledgments.- References.- 14 Rapid YAC End Sequencing by Alu-Vector PCR and Biotinylated Primers.- Summary.- 14.1 Introduction.- 14.2 Technical Procedures.- 14.3 Results and Discussion.- 14.4 Troubleshooting.- Acknowledgments.- References.
