Ligand blotting with 125I-fluoresceinamine-heparin |
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| Authors: | J W Smith D J Knauer |
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| Institution: | 1. Environmental Science Center, Qatar University, Doha, Qatar;2. Environmental Sciences Program, Department of Biological and Environmental Sciences, College of Arts and Sciences, Qatar University, PO Box 2713, Doha, Qatar;3. Quantitative Plant Ecology and Biodiversity Research Lab., Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran;1. Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai, 608 502, Tamil Nadu, India;2. Bioengineering and Drug Design Laboratory, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology, Chennai, 600036, Tamil Nadu, India;3. Department of Natural Resources and Waste Recycling, School of Energy, Environment and Natural Resources, Madurai Kamaraj University, Madurai, 625021, Tamil Nadu, India;1. Department of Plant Protection, Novosibirsk State Agrarian University, 630039, Novosibirsk, Russia;2. Faculty of Physical Engineering, Novosibirsk State Technical University, 630039, Novosibirsk, Russia;3. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630039, Novosibirsk, Russia;4. Zoology, Ryan Institute, School of Natural Sciences, University of Galway, Galway, H91 TK33, Ireland;5. Department of Biosciences, Faculty of Science and Engineering, Swansea University, Swansea, SA2 8PP, Wales, UK;1. Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan;2. Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, 1465-5 Aikawa, Kurume, Fukuoka 839-0861, Japan;1. Department of Microbiology, The University of Haripur, Haripur 22620 Pakistan;2. Department of Botany, PMAS-Arid Agriculture University Rawalpindi, Rawalpindi 46300 Pakistan;3. Department of General Sciences, Prince Sultan University, Rafha Street, Riyadh, Kingdom of Saudi Arabia;4. Department of Biotechnology, COMSATS University Islamabad, Abbottabad Campus, Abbottabad 22060, Pakistan;5. Department of Statistics & Mathematics, PMAS-Arid Agriculture University Rawalpindi, Rawalpindi 46300 Pakistan;6. Department of Agronomy, The University of Haripur, Haripur 22620 Pakistan |
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| 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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