Isolation, Purity Assessment, and Proteomic Analysis of Nuclei

The integrity of a subcellular proteomics is largely dependent on purity of the isolated compartment away from other contaminants. If high-purity nuclei is isolated, nuclear proteomics is a useful approach for investigating the mechanisms underlying plant physiological function. Although the isolation of high-purity nuclei from tissue or organ in plant is a difficult task, successful purification has been achieved through fractionation processes. For purification, there are five protocols such as (1) differential centrifugation, (2) discontinuous Percoll gradients, (3) continuous sucrose gradients, (4) combined continuous Percoll/sucrose gradients, and (5) continuous Percoll gradients. Furthermore, because purity assessment of purified nuclei is an important step, it is also described in this chapter.