By M. Dean Savage (auth.), Dr. Thomas Meier, Dr. Falk Fahrenholz (eds.)

ISBN-10: 3034873492

ISBN-13: 9783034873499

ISBN-10: 3034873514

ISBN-13: 9783034873512

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Additional info for A Laboratory Guide to Biotin-Labeling in Biomolecule Analysis

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Purification was performed in 3 sequential steps. First, antiserum was incubated with [3H]BANA-CCK -8s. The amount of nonspecific binding was determined by a parallel experiment where 10 flM pentagastrin was added at this stage. Alternatively, preimmune serum was used to distinguish specific from nonspecific binding. Then streptavidin-agarose beads were added, and the suspension was equilibrated for 3 hours at 4 DC to allow complete binding of the excess of biotinylated ligand and of the ligand-antibody complex to the matrix.

177,392-395. 29 Synthesis of Photocleavable Biotinylated Ligands and Application for Affinity Chromatography Christoph Thiele and Falk Fahrenholz Summary A method for affinity purification ba ed on the hi gh -affinity interaction of biotin-streptavidin and elution by irradiation with UV light was developed. As an examp le ofthi approach a biotinylated derivative of the peptide hormone cholecy tokinin (CCK-8s) containing a photocleavable o-nitrobenzyle ter group wa ynthesized. The ana log retained high affinity to both CCK receptor and antiCCK antibodies.

Biotinylation of sulfhydryl groups often provides an advantage in an application. For example, a protein in a complex mixture can be targeted for biotinylation if it is the only one with a free sulfhydryl group on its surface. IgG can be reduced under mild conditions, breaking the disulfide bond between the heavy chains while preserving the disulfide bond between the heavy and light chains, to form a monovalent fragment. The sulfhydryl of the reduced IgG is present at the hinge region and thus can be targeted for biotinylation at a site removed from the antigen-binding site.

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A Laboratory Guide to Biotin-Labeling in Biomolecule Analysis by M. Dean Savage (auth.), Dr. Thomas Meier, Dr. Falk Fahrenholz (eds.)

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