Heo Seok, Lubec Gert
Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria.
Electrophoresis. 2010 Dec;31(23-24):3789-96. doi: 10.1002/elps.201000374.
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.
尽管关于抗血清素受体1A（5HT1A_R）的一系列不同抗体已有报道，但关于这些抗体的特异性以及所识别抗原的信息却十分有限。在此，我们通过基于凝胶的蛋白质组学方法对一种抗体的反应性进行了表征，该方法应可作为未来定义抗体的一种模式。我们制备了一种针对5HT1A_R的抗体，并将其用于在含有5HT1A_R复合物的蓝色原胶上进行免疫沉淀和免疫印迹。5HT1A_R从组织中分离出来，并通过纳升液相色谱-电喷雾串联质谱（nano-LC-ESI-MS/MS）进行鉴定。在原胶上检测到一条单一的条带，在凝胶电泳的三维步骤中检测到一个代表变性受体的单一斑点。免疫沉淀显示变性的5HT1A_R有一条单一的条带。在此，我们提出了一种通过使用一种可靠的方法来表征抗体的程序，该方法可明确鉴定和表征来自小鼠全脑的抗原5HT1A_R。
