The use of conantokin and polyclonal antibodies to identify the NR2B subunit of the n-methyl-d-aspartate receptor
Journal Title: Revista MVZ Cordoba - Year 2010, Vol 15, Issue 3
Abstract
Objective. To propose a methodology that identifies the NR2B subunit through the use of conantokin G, as well as an adequate extraction of the NR2B subunit. Materials and methods. Two methodologies were tested for the extraction of the NR2B subunit of the adult rat, the first one sought the extraction of the subunit through a membrane by using sodium deoxicolate; and the second, guaranteed the solubilization and elimination of cytoplasmic proteins, in order to later extract the subunit through the use of the same detergent from the pellet resulting from the centrifugation of the obtained extract. Additionally, the conantokin G was biotynilated in order to evaluate its efficiency to identify the subunit and to compare the results obtained from traditional methodologies such as DOT-BLOT, WESTERN-BLOT, ELISA and Immunohistochemical. Results. The second methodology showed a greater extraction of NR2B, thus it was chosen for the subsequent extracts. The identification tests with biotynilated conantokin showed interference in recognition, thus, it was necessary identify the subunit NR2B through the use of the polyclonal antibodies mentioned in the tests. Conclusions. An impediment of esteric character is proposed in marking the conantokin with the biotin, which does not favor the interaction of this peptide with the subunit.
Authors and Affiliations
Edwin Reyes G, Lic. Química| Universidad Nacional de Colombia. Departamento de Química. Grupo de Investigación en Proteínas (GRIP). Cra 30 calle 45. Edificio 451. Laboratorio 201-1 Bogotá, D.C., Colombia., Edgar Reyes M, Ph. D *| Universidad Nacional de Colombia. Departamento de Química. Grupo de Investigación en Proteínas (GRIP). Cra 30 calle 45. Edificio 451. Laboratorio 201-1 Bogotá, D.C., Colombia.Correspondencia: eareyesm@unal.edu.co, Leonardo Lareo, Ph. D| Pontificia Universidad Javeriana, Departamento de Nutrición y Bioquímica, Bogotá, D.C., Colombia. †In Memoriam.
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