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.
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.
Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria.
Seok
S
Heo
Gert
G
Lubec
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D000918:Antibody Specificity; D015153:Blotting, Western / Q000379:methods*; D001923:Brain Chemistry; D002462:Cell Membrane / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D047468:Immunoprecipitation; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D040901:Proteomics; D044282:Receptor, Serotonin, 5-HT1A / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism
D000906:Antibodies; D010446:Peptide Fragments; D044282:Receptor, Serotonin, 5-HT1A