Barshop Institute for Longevity and Aging Studies and Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78240, USA.
Anal Biochem. 2011 Jun 15;413(2):123-32. doi: 10.1016/j.ab.2011.02.029. Epub 2011 Feb 24.
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.
秀丽隐杆线虫是一种模式生物，以其强大的遗传学而闻名。在线虫社区中，越来越需要将遗传学与生物化学相结合。然而，由于线虫厚厚的周围表皮，不使用强氧化变性剂或亚细胞区室从秀丽隐杆线虫中分离具有功能活性的蛋白质或核酸一直具有挑战性。Balch 匀浆机是一种在哺乳动物细胞培养生物学中得到广泛应用的工具。该仪器可互换的单个滚珠轴承设计允许细胞快速渗透或匀浆。在这里，我们展示了 Balch 匀浆机在秀丽隐杆线虫研究中的应用。我们描述了有效破碎和匀浆每个幼虫阶段（包括 dauer）的程序，并表明 Balch 匀浆机可用于提取具有功能活性的蛋白质。过氧化氢酶和二氢硫辛酸脱氢酶的酶促测定表明，使用 Balch 匀浆机进行样品制备与使用煮沸、超声或 Dounce 匀浆等传统方法相当或优于传统方法。我们还描述了无酚技术用于分离基因组 DNA 和 RNA。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 匀浆机是进行秀丽隐杆线虫生化研究的快速便捷解决方案。
