Jamieson-Lucy Allison, Mullins Mary C
Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Methods Mol Biol. 2019;1920:295-302. doi: 10.1007/978-1-4939-9009-2_17.
Proteomic characterization of isolated organelles can provide insight into the functional components of the structure and novel targets for further testing. Germplasm in developing oocytes is difficult to isolate for protein identification because not all types of germplasm are stable outside of the cytoplasm. In zebrafish, the Balbiani body forms a proteinaceous aggregate that contains the germplasm and we found is stable outside of the oocyte. Here we present a manual isolation protocol that collects intact Balbiani bodies from stage I zebrafish oocytes. We lysed oocytes by passing them through a syringe, and then used a fine injection needle to wick up Balbiani bodies by capillary action with minimal buffer solution. Using this protocol we collected sufficient material for proteomic analysis of the zebrafish Balbiani body.
对分离出的细胞器进行蛋白质组学表征，有助于深入了解该结构的功能成分以及为进一步测试提供新的靶点。发育中卵母细胞的种质难以分离用于蛋白质鉴定，因为并非所有类型的种质在细胞质外都稳定。在斑马鱼中，巴氏小体形成一种包含种质的蛋白质聚集体，我们发现其在卵母细胞外是稳定的。在此，我们展示一种手动分离方案，该方案可从I期斑马鱼卵母细胞中收集完整的巴氏小体。我们通过用注射器抽吸使卵母细胞裂解，然后用细注射针通过毛细作用吸取巴氏小体，所用缓冲液最少。使用该方案，我们收集到了足够的材料用于斑马鱼巴氏小体的蛋白质组学分析。
