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

Hardcover

ISBN 10: 376435206X ISBN 13: 9783764352066

Publisher: Birkhauser Verlag AG, 1996

Synopsis

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 ...

About this title

Synopsis

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.

Edition details

• Publisher: Birkhauser Verlag AG
• Publication date: 1996
• Language: English
• ISBN 10: 376435206X
• ISBN 13: 9783764352066
• Binding: Hardcover
• Number of pages: 254
• Editor: Meier Thomas, Fahrenholz Falk