ceptor-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 . . . . . . . . . . . . . . . . . . .

Contenuti

1 An Introduction to Avidin-Biotin Technology and Options for Biotinylation.- 1.1 Introduction.- The Avidin-Biotin Interaction.- Avidin.- Streptavidin.- Biotin.- 1.2 Technical Procedures.- Determining Biotin and Biotin Incorporation Levels.- Biotinylation Reagents.- General Considerations for Biotinylation.- Amine-Reactive Biotinylation Reagents.- Water-Soluble NHS Esters of Biotin.- Water-Insoluble NHS Esters of Biotin.- General Considerations for the Use of NHS-Ester Biotinylation Reagents.- Biotinylating IgG with NHS-Ester Biotinylation Reagents.- Carbohydrate-Directed Biotinylation Reagents.- Considerations for the Use of Hydrazide Derivatives of Biotin.- Carboxyl-Reactive Biotinylation Reagents.- Considerations for the Use of EDC.- Sulfhydryl-Reactive Biotinylation Reagents.- Considerations for the Use of Sulfhydryl-Reactive Biotinylation Reagents.- Biotinylation of Surface Thiols with Biotin-HPDP.- Biotinylating Reduced IgG with lodoacetyl-LC-Biotin.- Biotinylating Reduced IgG with Biotin-BMCC.- Phenylazide Derivatives of Biotin.- Considerations for the Use of Photoactivatable Biotin.- Biotinylation with Photoactivatable Biotin.- 1.3 Troubleshooting.- Avidin-HABA Biotin Assay.- NHS-Ester Biotinylation Reactions.- Biotin-Hydrazide Biotinylation Reactions.- EDC/5-(Biotinamido)pentylamine Reactions.- lodoacetyl-LC-Biotin Reactions.- Biotin-BMCC Reactions.- Phenylazide Biotinylation Reactions.- Acknowledgments.- References.- 2 Synthesis of Photocleavable Biotinylated Ligands and Application for Affinity Chromatography.- Summary.- 2.1 Introduction.- 2.2 Technical Procedures.- Chemical Synthesis.- 2.3 Results and Discussion.- Chemical Synthesis.- Binding to anti-CCK antisera and CCK-B receptors.- Binding to streptavidin agarose and photoelution.- Affinity chromatography.- 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.- Preparation of a Biotinylated Ligand.- Preparation of Membrane Fraction.- Solubilization of the PACAP Receptor.- Partial Purification of the PACAP Receptor.- Affinity Purification of the PACAP Receptor.- Final Purification of the PACAP Receptor.- Receptor-Binding Assay.- 3.3 Results and Discussion.- 3.4 Troubleshooting.- Impurity in PACAP27-Cys-NH2.- Low specific activity of the PACAP receptor despite a single band.- Acknowledgments.- References.- 4 Photoreactive Biotinylated Peptide Ligands for Affinity Labeling.- Summary.- 4.1 Introduction.- 4.2 Technical Procedures.- Synthesis of a trifunctional photoactivatable biotinylating reagent.- Synthesis of photoreactive biotinylated peptide hormones.- Site-specific incorporation of biotin and photo labels in separate steps.- Photoaffinity labeling.- 4.3 Results and Discussion.- Synthesis of photoactivatable insulins with permanent biotin labels.- Applications: Insulin.- Examples for other applications.- 4.4 Troubleshooting.- References.- 5 Immunoprecipitation of Biotinylated Cell Surface Proteins.- Summary.- 5.1 Introduction.- 5.2 Technical Procedures.- Cell lines.- 5.3 Results and Discussion.- 5.4 Troubleshooting.- Sample preparation.- Immunoprecipitation.- Western transfer, membranes, and detection.- Acknowledgments.- References.- 6 Biotinylation and Chemical Cross-Linking of Membrane Associated Molecules.- Summary.- 6.1 Introduction.- 6.2 Technical Procedures.- Chemicals and monoclonal antibodies.- Cell lines and preparation of cell suspensions.- 6.3 Results and Discussion.- Biotinylation and chemical cross-linking of molecules on the lymphocyte surface.- Permeabilizing cells with lysolecithin to biotinylate and cross-link intracellular molecules.- 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.- Biotinylation of lectins.- The microtiter plate assay.- Lectinoblotting.- 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.- Purification of poly(ADP-ribose) polymerase and preparation of poly (ADP-ribose).- Biotinylation of poly(ADP-ribose).- Use of biotinylated poly(ADP-ribose) for ligand blotting.- 8.3 Results and Discussion.- 8.4 Troubleshooting.- Purification and storage of poly(ADP-ribose) polymerase.- Preparation of poly(ADP-ribose).- Unspecific signals at ligand blotting.- Time considerations.- 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.- Biotinylation procedures.- Compositional and functional analyses conducted on the biotinylated probes.- Calculation of IC50 values.- Applications of the biotinylated probes.- 9.3 Results and Discussion.- Biotinylated SLPI.- Biotinylated PCTI-1.- Biotinylated aprotinin.- Biotinylated trypsin.- 9.4 Troubleshooting.- General considerations.- Specific considerations.- 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.- Modification of RBC with biotin/streptavidin and attachment of b-IgG to RBC.- Stability of immunoerythrocytes in serum: in vitro study.- Binding of immunoerythrocytes to antigen: in vitro study.- Biodistribution and circulation of serum-stable immunoerythrocytes in rats.- Conclusion.- 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.- General.- 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.- PCR-labeling of (c)DNA probes.- Northern blot.- Southern blot.- Quantitative analysis by densitometry.- 12.3 Results and discussion.- 12.4 Troubleshooting.- No or weak signal.- Background problems.- References.- 13 Biotin-Labeled Riboprobes to Study RNA-Binding Proteins.- Summary.- 13.1 Introduction.- 13.2 Technical Procedures.- Preparation of biotinylated riboprobes.- 13.3 Results and Discussion.- 13.4 Troubleshooting.- Biotinylated riboprobes.- Northwestern assay.- Acknowledgments.- References.- 14 Rapid YAC End Sequencing by Alu-Vector PCR and Biotinylated Primers.- Summary.- 14.1 Introduction.- 14.2 Technical Procedures.- Isolation of YAC DNA.- Alu-vector PCR.- 14.3 Results and Discussion.- 14.4 Troubleshooting.- Acknowledgments.- References.

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