Development of a Fluorescence Titration Method to Measure the Association Constants of Small Molecule Fluorophores with Cyclodextrins and Fluorophore Specific Antibodies
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Authors
Meyers, Jessica R.
Issue Date
2006
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
In this study, a protocol was developed to measure the association constant (Ks) of a fluorophore and a non-fluorescent binding protein using fluorescence titration techniques. A spectrofluorimeter was used to measure the fluorescence intensity of a solution during the titration of two binding partners, and by using non-linear regression techniques, the Ks was measured. One of the binding partners must be fluorescent and result in a change in its emission spectrum upon binding to a macromolecular binding
host. The fluorophores 8-anilino-l-naphthalenesulfonate (ANS) and
dimethylaminonaphthalene sulfonyl (dansyl) amide were used as small molecule ligands, and P-cyclodextrin (BCD), hydroxypropyl-P-cyclodextrin (HPBCD), and an antibody against dansyl (anti-DAN) were used as macromolecular binding hosts. A method for measuring and calculating Ks was developed for four pairs of binding partners: ANS:BCD, dansyl:BCD, ANS:HPBCD, and dansyl:anti-DAN. This method involves the titration of one binding partner into the other at specified concentration ranges, and a
non-linear regression equation for calculating the Ks. This protocol can be used for measuring the association constant of these fluorescent ligands and any other macromolecular binding host.
Description
viii, 95 p.
Citation
Publisher
Kalamazoo College
License
U.S. copyright laws protect this material. Commercial use or distribution of this material is not permitted without prior written permission of the copyright holder. All rights reserved.