Isolation of Nuclear Proteins

Abstract

Proteome analysis is becoming a powerful tool of discovery-driven research, with investigations ranging from whole organisms to specific subcellular compartments. Especially for the latter, efficient and robust methods for protein purification are the prerequisite for obtaining meaningful proteomic data. The plant nucleus is the repository of critical components of the genetic and biochemical machinery and therefore of great interest as source material for proteomic studies. Although Arabidopsis thaliana is the prime model system for plant molecular genetics, proven protocols for biochemical fractionation are often best established for other plant species. In this chapter, we present methods for the isolation of nuclei, nuclear proteins, and nuclear protein fractions that have been adapted for Arabidopsis. Two different protocols for the isolation of nuclei and nuclear extracts from Arabidopsis plants and suspension cells are described. Discovering that developmental mutant phenotypes are based on chromatin-associated and chromatin-altering proteins has spiked a growing interest in Arabidopsis chromatin proteins. We describe a method for obtaining an Arabidopsis protein chromatin fraction and the entire histone complement.

The Arabidopsis immune adaptor SRFR1 interacts with TCP transcription factors that redundantly contribute to effector‐triggered immunity

“…For transgenic Arabidopsis expressing localization‐tagged HA–SRFR1, cellular fractionation was performed on plant tissue from liquid cultures based on a modified protocol adapted from Calikowski and Meier () by Ying Wan and Scott Peck (Division of Biochemistry, University of Missouri, Columbia, MO). Briefly, tissue was processed in nuclei isolation buffer (13.8% hexylene glycol, 20 m m β‐mercaptoethanol, 50 μ m spermine, 125 μ m spermidine, 20 m m KCl, 20 m m HEPES, pH 7.4) containing 0.3% Triton and protease and proteasome inhibitors (Sigma).…”

Shotgun proteomic analysis of Arabidopsis thaliana leaves

“…For the specific enrichment of proteins, we purified nuclei, microsomal membranes, and plastids and mitochondria [10,11]. For nuclei, frozen leaf powders were filtered through several membranes.…”

Removal of high‐abundance proteins for nuclear subproteome studies in rice (Oryza sativa) endosperm

“…A flow rate of 3 mL/min was used for both SCX and RP columns. For SCX, a salt step-gradient of 0,10,15,25,30,35,40,45, 50, 57, 64, 90, and 700 mM ammonium acetate in 5% ACN and 0.1% formic acid were applied. The eluted peptides were loaded directly on the RP column, equilibrated with 0.1% formic acid and 5.0% ACN.…”