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Use of a Monoclonal Antibody to Purify the Tetrodotoxin Binding Component from the Electroplax of Electrophorus electricus

Nakayama, Hitoshi and Withy, Raymond M. and Raftery, Michael A. (1982) Use of a Monoclonal Antibody to Purify the Tetrodotoxin Binding Component from the Electroplax of Electrophorus electricus. Proceedings of the National Academy of Sciences of the United States of America, 79 (25). pp. 7575-7579. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:NAKpnas82

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Abstract

The tetrodotoxin binding component of the voltage-sensitive sodium channel from Electrophorus electricus electroplax was purified by using a monoclonal antibody. An impure preparation of tetrodotoxin binding component was mixed with the pure monoclonal antibody, and the immune complex so formed was isolated by affinity chromatography on a protein A-Sepharose column. Excess antibody was removed by ion-exchange chromatography. The purified material has a specific activity of over 1,800 pmol of [3H]tetrodotoxin bound per mg of protein. By assuming that the immune complex has a stoichiometry of 1:1, this specific activity then represents an actual specific activity of 3,000 pmol of [3H]tetrodotoxin bound per mg of eel electroplax protein, or 75% of the theoretical specific activity expected for a pure toxin binding component of Mr 250,000. The peptide composition of the purified material was simple with the predominant species present being of Mr approx 250,000. Minor components were also present with M{rs} of approx 95,000, approx 44,000, and approx 23,000.


Item Type:Article
Additional Information:Copyright © 1982 by the National Academy of Sciences Communicated by Ray D. Owen, August 5, 1982 We thank Drs. Jeremy P. Brockes, Larry Fritz, and Katie Stygall for helpful discussions concerning many aspects ofthis work and for the gifts of immunoreagents. In addition, we thank Dr. Larry Fritz for growing VD10 in culture and helping with ascites fluid production. Laurie Jutzi typed the manuscript, and Valerie Purvis produced the artwork. This research was supported by U.S. Public Health Service Grants NS 12018 and GM 16424 and by a grant from the Pew Charitable Trust. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Subject Keywords:action potential; neurotoxin binding; protein A; sodium channel
Record Number:CaltechAUTHORS:NAKpnas82
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:NAKpnas82
Alternative URL:http://www.pnas.org/cgi/content/abstract/79/23/7575
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:5791
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:02 Nov 2006
Last Modified:14 Nov 2014 19:19

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