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A Laboratory Guide to Biotin-Labeling in Biomolecule Analysis: (v. 7 Biomethods)

A Laboratory Guide to Biotin-Labeling in Biomolecule Analysis: (v. 7 Biomethods)

          
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About the Book

This book summarizes protocols and applications of recently developed or improved non-radioactive biotin-labelling techniques for proteins, glycoproteins and nucleic acids and will provide valuable help to researchers both in fundamental and in applied sciences. The first chapter of this volume compares the chemical properties of biotin-labelling compounds currently available and outlines their reactions principles. The following contributions provides a step-by-step protocol on how to prepare and successfully apply biotin-labelled probes for the analysis of complex biochemical and cellular systems. An extended troubleshooting section completes each of the protocols. In most cases these core protocols provide a guideline that encourage modifications according to the researchers' experimental designs. Combined with sensitive detection, these recently developed experimental procedures are powerful tools for many applications in areas ranging from protein biochemistry to molecular and cellular biology.

Table of Contents:
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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Product Details
  • ISBN-13: 9783764352066
  • Publisher: Birkhauser Verlag AG
  • Publisher Imprint: Birkhauser Verlag AG
  • Height: 240 mm
  • No of Pages: 237
  • Weight: 700 gr
  • ISBN-10: 376435206X
  • Publisher Date: 01 Feb 1996
  • Binding: Hardback
  • Language: English
  • Series Title: v. 7 Biomethods


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