Max Planck Institute for Chemical Ecology, Jena, Germany.
Anal Biochem. 2010 Mar 15;398(2):198-202. doi: 10.1016/j.ab.2009.11.016. Epub 2009 Nov 14.
We have established a method for the isolation of chloroplasts from Arabidopsis thaliana that allows proteomic studies in the context of biotic stress with small amounts of starting material. Employing a 50% Percoll layer to separate crude filtrates, the required leaf material was reduced to 2-3g, yielding more than 300 microg of chloroplast proteins. The quality of this fraction was confirmed by immunological, enzymatic, and gel-based assays. This protocol provides intact chloroplasts from Arabidopsis plants with a high degree of integrity and purity as well as sufficient protein recovery, thereby enabling studies of plant-herbivore or plant-pathogen interactions.
我们建立了一种从拟南芥中分离叶绿体的方法，该方法可在生物胁迫的情况下，使用少量起始材料进行蛋白质组学研究。采用 50%Percoll 层分离粗滤液，所需叶片材料减少至 2-3g，可获得超过 300μg 的叶绿体蛋白。该级分的质量通过免疫学、酶学和基于凝胶的测定得到证实。该方案提供了具有高度完整性和纯度的完整叶绿体以及足够的蛋白质回收，从而能够研究植物-草食动物或植物-病原体相互作用。
