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Generation and characterization of a specific polyclonal antibody against the mouse serotonin receptor 1A: A state‐of‐the‐art recommendation on how to characterize antibody specificity
Author(s) -
Heo Seok,
Lubec Gert
Publication year - 2010
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201000374
Subject(s) - immunoprecipitation , polyclonal antibodies , antibody , antigen , microbiology and biotechnology , gel electrophoresis , receptor , 5 ht receptor , chemistry , serotonin , computational biology , biology , biochemistry , immunology
A series of different antibodies against serotonin receptor 1A (5HT1A_R) have been reported although only limited information on the specificity of these antibodies and the antigens recognized is available. Herein, we characterized reactivity of an antibody by a gel‐based proteomics method that should represent a model how antibodies may be defined in the future. An antibody against the 5HT1A_R was generated, used for immunoprecipitation and immunoblotting on blue‐native gels containing a 5HT1A_R complex. The 5HT1A_R was isolated from tissue and was defined by nano‐LC‐ESI‐MS/MS. A single band on the native gel and a single spot representing the denatured receptor in the 3rd dimensional step of gel electrophoresis was detected. Immunoprecipitation revealed a single band for the denatured 5HT1A_R. Herein, a procedure is proposed to characterize an antibody by the use of a robust method unambiguously identifying and characterizing the antigen, 5HT1A_R, from mouse whole brain.