Fluorescence polarization assay
a fluorescence polarization and assay technology, applied in the field of fluorescence polarization assay, can solve the problems of limited antibody-based molecular recognition molecules, and therefore useful in diagnostic assays, and the platform that has typically been used to identify and quantify aptamers bound to analytes is very limited
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[0091] A. Methods for assessing binding of proteins to aptamer-coupled beads
[0092] The polarization anisotropy of dye-labeled aptmers changes with time upon binding to a specific protein. Changes in fluorescence anisotropy of 3 aptamers bound to a solid support upon binding of specific proteins was measured.
[0093] The three fluorophore labeled aptamers used in these experiments were anti-thrombin aptamer (FAM-labeled, a 15 mer), which is specific for thrombin from human plasma, 518-aptamer (FAM-labeled, a 60 mer), which is specific for GP120MN, an HIV-1 protein and 650-aptamer (FAM-labeled, a 60 mer), which is specific for recombinant human FGF basic protein.
[0094] 1. Anti-Thrombin Aptamer for Thrombin
[0095] Binding of thrombin to FAM-labeled anti-thrombin aptamers was measured. 5 micron silica particles were purchased from Bangs Laboratories (Ohio, USA) and pretreated with 1N NaCI solution at 100° C. for 1 hour. The silica particles were then treated for 30 minutes with 3-amino...
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