Izquierdo Yovanny, Fernández-Santos Rubén, Cascón Tomás, Castresana Carmen
Department of Plant Molecular Genetics, Centro Nacional de Biotecnología, Madrid, Spain.
Bio Protoc. 2020 Dec 20;10(24):e3867. doi: 10.21769/BioProtoc.3867.
Lipid droplets (LDs) are neutral lipid aggregates surrounded by a phospholipid monolayer and specific proteins. In plants, they play a key role as energy source after seed germination, but are also formed in vegetative tissues in response to developmental or environmental conditions, where their functions are poorly understood. To elucidate these, it is essential to isolate LDs with good yields, while retaining their protein components. LD isolation protocols are based on their capacity to float after centrifugation in sucrose gradients. Early strategies using stringent conditions and LD-abundant plant tissues produced pure LDs where core proteins were identified. To identify more weakly bound LD proteins, recent protocols have used low stringency buffers, but carryover contaminants and low yields were often a problem. We have developed a sucrose gradient-based protocol to isolate LDs from Arabidopsis leaves, using Tween-20 and fresh tissue to increase yield. In both healthy and bacterially-infected Arabidopsis leaves, this protocol allowed to identify LD proteins that were later confirmed by microscopy analysis.
脂滴（LDs）是由磷脂单层和特定蛋白质包围的中性脂质聚集体。在植物中，它们在种子萌发后作为能量来源发挥关键作用，但在营养组织中也会因发育或环境条件而形成，其功能尚不清楚。为了阐明这些功能，必须以高产率分离脂滴，同时保留其蛋白质成分。脂滴分离方案基于其在蔗糖梯度中离心后漂浮的能力。早期使用严格条件和富含脂滴的植物组织的策略产生了纯脂滴，其中鉴定出了核心蛋白。为了鉴定更多弱结合的脂滴蛋白，最近的方案使用了低严格度缓冲液，但残留污染物和低产率常常是个问题。我们开发了一种基于蔗糖梯度的方案，使用吐温 - 20 和新鲜组织从拟南芥叶片中分离脂滴，以提高产量。在健康和细菌感染的拟南芥叶片中，该方案都能够鉴定出脂滴蛋白，这些蛋白随后通过显微镜分析得到了证实。
