A Method for Extracting the Nuclear Scaffold from the Chromatin Network.

Abstract

Each cell contains many large DNA polymers packed in a nucleus of approx. 10 μm in diameter. With histones, these DNA polymers are known to form chromatins. How chromatins further compact in the nucleus is unclear but it inevitably depends on an extensive non-chromatin nuclear scaffold. Imaging of endogenous chromatin network and the complementary scaffold that support this network has not been achieved but biochemical and proteomic investigations of the scaffold can still provide important insights into this chromatin-organizing network. However, this demands highly inclusive and reproducible extraction of the nuclear scaffold. We have recently developed a simple protocol for releasing the scaffold components from chromatins. The inclusiveness of the extract was testified by the observation that, upon its extraction from the nuclei, the remaining nuclear chromatins were liberated into extended and often parallel chromatin fibers. Basically, this protocol includes the generation of pure nuclei, treatment of the nuclei with Triton X-100 to generate envelope-depleted nuclei (TxN), and extraction of the nuclei at 500 mM NaCl in a sucrose-containing buffer. This combined extract of TxN is known as TxNE.