Protocol

Organelle Proteomics

Volume 432 of the series Methods in Molecular Biology™ pp 139-148

Purification and Proteomic Analysis of a Nuclear-Insoluble Protein Fraction

  • Tsuneyoshi HorigomeAffiliated withDepartment of Chemistry, Faculty of Science, Niigata University
  • , Kazuhiro FurukawaAffiliated withDepartment of Chemistry, Faculty of Science, Niigata University
  • , Kohei IshiiAffiliated withDepartment of Chemistry, Faculty of Science, Niigata University

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Summary

We describe here a method for analyzing a rat liver nuclear-insoluble protein fraction to determine candidate proteins participating in nuclear architecture formation. Rat liver nuclei are purified by sucrose density gradient centrifugation. The purified nuclei are treated with DNase and RNase and then washed with high salt and detergent solutions. The residual nuclear-insoluble protein fraction is separated by reversed-phase high-performance liquid chromatography (HPLC) in 60% formic acid on a polystyrene resin column. This system allows good resolution and high recovery of most insoluble proteins, including intrinsic membrane proteins and even proteins larger than 140 kDa, with more than 70% recovery. The LC-fractionated proteins are further separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Protein bands are excised, in-gel digested with trypsin, and then analyzed with a protein sequencer or mass spectrometer. Using this protocol, 138 were separated, 29 were identified, among which one appears as a novel nuclear constituent localized in the interchromatin space.

Key Words

Nuclear-insoluble protein nuclear proteome subcellular fractionation polymer-based column reversed-phase HPLC membrane protein purification perfusion chromatography