Fractionation and Extraction of Crude Nuclear Proteins From Arabidopsis Seedlings

Background

The plant nucleus is the repository of a cell that regulates the genetic and biochemical machinery, therefore it is crucial to isolate the plant nuclei as source material for proteomic studies, protein expression patterns, modifications, and dynamic physiological responses (Calikowski and Meier, 2006). Nuclei isolation from different plant tissues mainly involves four steps, namely disruption, filtration, repeated centrifugation, and density gradient separation (Yin and Komatsu, 2016). Homogenization buffers are important for the stabilization of nuclei from various plant tissues. The compositions of homogenization buffers includes organic buffers (e.g., MES, HEPES, PIPES, and Tris) to stabilize the pH, inorganic salts (e.g., KCl, NaCl, and MgCl₂) to maintain the ionic strength of the solution, sucrose, glycerol, and hexylene glycol to stabilize membranes, polyamines (spermine and spermidine) to stabilize the nuclear chromatin and avoid the aggregation of nuclei, reducing agents (β-mercaptoethanol and dithiothreitol) to maintain cysteine residues in the reduced form, and protease inhibitors (phenylmethylsulfonyl fluoride and plant protease inhibitor cocktail) to protect proteins from degradation (Yin and Komatsu, 2016). For the fact that various organelles of a cell differ in both size and density, density gradient centrifugation (also known as rate-zonal centrifugation) and differential-velocity centrifugation are widely used for the purification of different subcellular organelles (Komatsu, 2007). Percoll density gradient is often used in the fractionation and purification of nuclei from cell homogenates of Arabidopsis seedlings (Calikowski and Meier, 2006). The 2.0 M sucrose gradient method is also rapid and efficient for the isolation of nuclei from the cultured rice suspension cells (Morré and Andersson, 1994). Here, we present a simple protocol for the fractionation of the crude nuclei from Arabidopsis seedlings without density gradient centrifugation, based on the previously established methods from Calikowski and Meier (2006) and Liu et al. (2018). This method is suitable for analyzing accumulation, as well as nuclear and cytoplasmic distributions of specific proteins under specific stress conditions (Wang et al., 2011; Liu et al., 2018; Xu et al., 2021). However, percoll density gradient centrifugation is recommended, if high-quality nuclear proteins are needed.