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

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Abstract

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.

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