Breaking Caenorhabditis elegans the easy way using the Balch homogenizer: An old tool for a new application
Breaking Caenorhabditis elegans the easy way using the Balch homogenizer: An old tool for a new application
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DOI：
10.1016/j.ab.2011.02.029
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发表时间：
2011-06-15
影响因子：
2.9
通讯作者：
Rea, Shane L.
中科院分区：
文献类型：
--
作者：
Bhaskaran, Shylesh;Butler, Jeffrey A.;Rea, Shane L.
线虫秀丽隐杆线虫是一种以其强大的遗传学而闻名的模式生物。蠕虫界越来越需要将遗传学与生物化学结合起来。然而，在不使用强氧化变性剂或亚细胞区室的情况下从线虫中分离功能活性蛋白质或核酸一直具有挑战性，因为蠕虫的厚的周围角质层。Balch匀浆器是一种在哺乳动物细胞培养生物学中有很大用途的工具。该仪器的可互换单滚珠轴承设计允许细胞的快速透化或均质化。在这里，我们展示了实用的Balch匀浆器与线虫的研究。我们描述了每个幼虫阶段，包括dauers有效的破碎和均质化的程序，并表明，Balch匀浆器可用于提取功能活性蛋白。过氧化氢酶和二氢硫辛酰胺脱氢酶的酶测定表明，使用Balch匀浆器的样品制备等于或优于采用煮沸，超声处理或Dounce均质化的常规方法。我们还描述了用于分离基因组DNA和RNA的无酚技术。最后，我们使用该工具分离偶联的线粒体和多核糖体。可重复使用的Balch均质器代表了对线虫进行生化研究的快速方便的解决方案。(C)2011 Elsevier Inc. All rights reserved.
The nematode Caenorhabditis elegans is a model organism best known for its powerful genetics. There is an increasing need in the worm community to couple genetics with biochemistry. Isolation of functionally active proteins or nucleic acids without the use of strong oxidizing denaturants or of subcellular compartments from C elegans has, however, been challenging because of the worms' thick surrounding cuticle. The Balch homogenizer is a tool that has found much use in mammalian cell culture biology. The interchangeable single ball-bearing design of this instrument permits rapid permeabilization, or homogenization, of cells. Here we demonstrate the utility of the Balch homogenizer for studies with C elegans. We describe procedures for the efficient breakage and homogenization of every larval stage, including dauers, and show that the Balch homogenizer can be used to extract functionally active proteins. Enzymatic assays for catalase and dihydrolipoamide dehydrogenase show that sample preparation using the Balch homogenizer equals or outperforms conventional methods employing boiling, sonication, or Dounce homogenization. We also describe phenol-free techniques for isolation of genomic DNA and RNA. Finally, we used the tool to isolate coupled mitochondria and polysomes. The reusable Balch homogenizer represents a quick and convenient solution for undertaking biochemical studies on C elegans. (C) 2011 Elsevier Inc. All rights reserved.