Fluorometric assay for avidin—biotin interaction

Donald M. Mock, Paul Horowitz

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

The interaction of avidin and biotin can be quantitated by measuring the fluorescence changes occurring when the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid (2, 6-ANS) is displaced by biotin from the biotin-binding site on avidin. The probe binds to a hydrophobic region at or near the biotin-binding site on avidin. The wavelength of maximum intensity depends on the solvent system in which the probe is dissolved. Because the equilibrium dissociation constant is about 200 μM, neither the bound nor the free probe concentrations are negligible with respect to the other at the concentrations typically used. The wavelengths are chosen to maximize the difference in fluorescence intensity between bound and free 2,6-ANS based on published maxima for excitation and emission while avoiding scattered light from the excitation beam. Initially, avidin and 2,6-ANS are added to a quartz cuvette, mixed, and placed in the fluorometer. The maximum fluorescence enhancement is reached in the few seconds required for mixing and insertion of the cuvette; no extra incubation time is required at this point or during the titration.

Original languageEnglish (US)
Pages (from-to)234-240
Number of pages7
JournalMethods in Enzymology
Volume184
Issue numberC
DOIs
StatePublished - Jan 1 1990
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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