Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, 27695-7905, USA.
Department of Biological Sciences, North Carolina State University, Raleigh, NC, 27695-7614, USA.
Sci Rep. 2021 Mar 18;11(1):6323. doi: 10.1038/s41598-021-85923-x.
UBE3A is an E3 ubiquitin ligase encoded by the neurally imprinted UBE3A gene. The abundance and subcellular distribution of UBE3A has been the topic of many previous studies as its dosage and localization has been linked to neurodevelopmental disorders including Autism, Dup15q syndrome, and Angelman syndrome. While commercially available antibodies have been widely employed to determine UBE3A localization, an extensive analysis and comparison of the performance of different UBE3A antibodies has not been conducted. Here we evaluated the specificities of seven commercial UBE3A antibodies in two of the major experimental models used in UBE3A research, mouse and human pluripotent stem cell-derived neural cells and tissues. We tested these antibodies in their two most common assays, immunofluorescence and western blot. In addition, we also assessed the ability of these antibodies to capture dynamic spatiotemporal changes of UBE3A by utilizing human cerebral organoid models. Our results reveal that among the seven antibodies tested, three antibodies demonstrated substantial nonspecific immunoreactivity. While four antibodies show specific localization patterns in both mouse brain sections and human cerebral organoids, these antibodies varied significantly in background signals and staining patterns in undifferentiated human pluripotent stem cells.
UBE3A 是一种 E3 泛素连接酶，由神经印迹的 UBE3A 基因编码。UBE3A 的丰度和亚细胞分布一直是许多先前研究的主题，因为其剂量和定位与神经发育障碍有关，包括自闭症、Dup15q 综合征和 Angelman 综合征。虽然商业上可获得的抗体已被广泛用于确定 UBE3A 的定位，但尚未对不同 UBE3A 抗体的性能进行广泛的分析和比较。在这里，我们评估了在 UBE3A 研究中使用的两种主要实验模型（小鼠和人多能干细胞衍生的神经细胞和组织）中的七种商业 UBE3A 抗体的特异性。我们在其最常见的两种检测方法，免疫荧光和 Western blot 中测试了这些抗体。此外，我们还评估了这些抗体通过利用人类大脑类器官模型捕捉 UBE3A 的动态时空变化的能力。我们的结果表明，在测试的七种抗体中，三种抗体显示出大量的非特异性免疫反应。虽然四种抗体在小鼠脑切片和人类大脑类器官中均显示出特定的定位模式，但这些抗体在未分化的人多能干细胞中的背景信号和染色模式存在显著差异。
