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Ligand blotting with125I-fluoresceinamine-heparin

Authors
Journal
Analytical Biochemistry
0003-2697
Publisher
Elsevier
Publication Date
Volume
160
Issue
1
Identifiers
DOI: 10.1016/0003-2697(87)90619-1
Keywords
  • Heparin
  • Protease Nexin-I
  • Antithrombin Iii
  • Ligand Blotting
  • Cleveland Digestion
  • Fluoresceinamine
Disciplines
  • Biology

Abstract

Abstract A highly sensitive method for ligand blotting with heparin has been developed. This ligand-blotting method is successful largely due to the ability to prepare heparin derivatives of high radiospecific activity. Heparin was modified with fluoresceinamine according to the method of C. G. Glabe, P. K. Harty, and S. D. Rosen ((1983) Anal. Biochem. 130, 287–294), and this fluoresceinamine-derivatized heparin can be radioiodinated to a specific activity of 100,000 cmp/ng of uronic acid. This is a 500-fold increase in specific activity over Bolton-Hunter-modified heparin, as prepared by A. D. Cardin, K. R. Witt, and R. L. Jackson ((1984) Anal. Biochem. 137, 368–373). 125I-Fluoresceinamine-derivatized heparin retains its ability to interact specifically with heparin-binding proteins such as human protease nexin-I and antithrombin III. 125I-Fluoresceinamine-derivatized heparin can be used to visualize and quantify heparin binding proteins on nitrocellulose. Protease nexin-I can be visualized at the nanogram level. In addition, ligand blotting with 125I-fluoresceinamine heparin can be combined with Cleveland digestion ( D. W. Cleveland, S. Fisher, M. W. Kirschner, and U. K. Laemmli (1977) J. Biol. Chem. 252, 1102–1106) in order to identify heparin binding fragments of proteins with heparin binding domains.

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