Evaluation of UBE3A antibodies in mice and human cerebral organoids

Abstract

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.

Regional and cellular organization of the autism-associated protein UBE3A/E6AP and its antisense transcript in the brain of the developing rhesus monkey

“…To label UBE3A, we selected the mouse monoclonal antibody 3E5 (Sigma-Aldrich Cat# SAB1404508, RRID:AB_10740376). This widely used antibody offers several advantages, including extensive validation ( Judson et al, 2014 ; Burette et al, 2017 ; Sen et al, 2021 ). As a monoclonal antibody, it offers the advantage of being producible in virtually unlimited quantities with consistent properties, ensuring consistency and reproducibility across experiments over extended periods.…”

The porcine islet-derived organoid showed the characteristics as pancreatic duct

“…It is an in vitro a three-dimensional (3D) cellular cluster derived from primary tissue or pluripotent stem cells with the capabilities of self-renewal, self-organization, and similar organ functionality 3 . Since today, Various organoids which harbor the characteristics of each organ including brain 4 , lung 5 , liver 6 , thyroid 7 have been widely developed. Regarding pancreatic islet, some groups succeeded to develop islet organoid which ameliorated diabetic animals.…”