Skip to main content
SpringerLink
Log in

Isolation, Purity Assessment, and Proteomic Analysis of Nuclei

  • Protocol
  • First Online:
Plant Membrane Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1696))

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 159.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Yin X, Komatsu S (2016) Plant nuclear proteomics for unraveling physiological function. New Biotechnol 33:644–654

    Article  CAS  Google Scholar 

  2. Wilson KL, Dawson SC (2011) Functional evolution of nuclear structure. J Cell Biol 195:171–181

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Aebersold R, Mann M (2003) Mass spectrometry based proteomics. Nature 422:198–207

    Article  CAS  PubMed  Google Scholar 

  4. Petrovská B, Šebela M, Doležel J (2015) Inside a plant nucleus: discovering the proteins. J Exp Bot 66:1627–1640

    Article  PubMed  Google Scholar 

  5. Pandey A, Choudhary MK, Bhushan D, Chattopadhyay A, Chakraborty S, Datta A et al (2006) The nuclear proteome of chickpea (Cicer arietinum L.) reveals predicted and unexpected proteins. J Proteome Res 5:3301–3311

    Article  CAS  PubMed  Google Scholar 

  6. Loureiro J, Rodriguez E, Doležel J, Santos C (2007) Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species. Ann Bot 100:875–888

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sikorskaite S, Rajamäki ML, Baniulis D, Stanys V, Valkonen JP (2013) Protocol: optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families. Plant Methods 9:31

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. McKeown P, Pendle AF, Shaw PJ (2008) Preparation of Arabidopsis nuclei and nucleoli. In: Hancock R (ed) The nucleus, vol 1. Humana Press, New York, pp 67–75

    Chapter  Google Scholar 

  9. Bae MS, Cho EJ, Choi EY, Park OK (2003) Analysis of the Arabidopsis nuclear proteome and its response to cold stress. Plant J 36:652–663

    Article  CAS  PubMed  Google Scholar 

  10. Aki T, Yanagisawa S (2009) Application of rice nuclear proteome analysis to the identification of evolutionarily conserved and glucose-responsive nuclear proteins. J Proteome Res 8:3912–3924

    Article  CAS  PubMed  Google Scholar 

  11. Kumar R, Kumar A, Subba P, Gayali S, Barua P, Chakraborty S et al (2014) Nuclear phosphoproteome of developing chickpea seedlings (Cicer arietinum L.) and protein-kinase interaction network. J Proteome 105:58–73

    Article  CAS  Google Scholar 

  12. Guo B, Chen Y, Li C, Wang T, Wang R, Wang B et al (2014) Maize (Zea mays L.) seedling leaf nuclear proteome and differentially expressed proteins between a hybrid and its parental lines. Proteomics 14:1071–1087

    Article  CAS  PubMed  Google Scholar 

  13. Choudhary MK, Basu D, Datta A, Chakraborty N, Chakraborty S (2009) Dehydration-responsive nuclear proteome of rice (Oryza sativa L.) illustrates protein network, novel regulators of cellular adaptation, and evolutionary perspective. Mol Cell Proteomics 8:1579–1598

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Komatsu S (2007) Extraction of nuclear proteins. Methods Mol Biol 355:73–77

    CAS  PubMed  Google Scholar 

  15. Khan MM, Komatsu S (2004) Rice proteomics: recent developments and analysis of nuclear proteins. Phytochemistry 65:1671–1681

    Article  CAS  PubMed  Google Scholar 

  16. Li M, Yin X, Sakata K, Yang P, Komatsu S (2015) Proteomic analysis of phosphoproteins in the rice nucleus during early stage of seed germination. J Proteome Res 14:2884–2896

    Article  CAS  PubMed  Google Scholar 

  17. Komatsu S, Han C, Nanjo Y, Altaf-Un-Nahar M, Wang K, He D, Yang P (2013) Label-free quantitative proteomic analysis of abscisic acid effect in early-stage soybean under flooding. J Proteome Res 12:4769–4784

    Article  CAS  PubMed  Google Scholar 

  18. Komatsu S, Hiraga S, Nouri MZ (2014) Analysis of flooding-responsive proteins localized in the nucleus of soybean root tips. Mol Biol Rep 41:1127–1139

    Article  CAS  PubMed  Google Scholar 

  19. Oh MW, Nanjo Y, Komatsu S (2014) Identification of nuclear proteins in soybean under flooding stress using proteomic technique. Protein Pept Lett 21:458–467

    Article  CAS  PubMed  Google Scholar 

  20. Yin X, Komatsu S (2015) Quantitative proteomics of nuclear phosphoproteins in the root tip of soybean during the initial stages of flooding stress. J Proteome 119:183–195

    Article  CAS  Google Scholar 

  21. Yin X, Komatsu S (2016) Nuclear proteomics reveals the role of protein synthesis and chromatin structure in root tip of soybean during the initial stage of flooding stress. J Proteome Res 15:2283–2298

    Article  CAS  PubMed  Google Scholar 

  22. Komatsu S, Nanjo Y, Nishimura M (2013) Proteomic analysis of the flooding tolerance mechanism in mutant soybean. J Proteome 79:231–250

    Article  CAS  Google Scholar 

  23. Kato M, Shimizu S (1987) Chlorophyll metabolism in higher plants: VII. Chlorophyll degradation in senescing tobacco leaves; phenolic-dependent peroxidative degradation. Can J Bot 65:729–735

    Article  CAS  Google Scholar 

  24. Huang S, Jacoby RP (1999) Plant mitochondrial proteomics. In: Millar AH, Taylor NL (eds) Plant proteomics. Humana Press, New York, pp 499–524

    Google Scholar 

  25. Hasinoff BB (1990) Inhibition and inactivation of NADH-cytochrome c reductase activity of bovine heart submitochondrial particles by the iron (III)-adriamycin complex. Biochem J 265:865–870

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Yin X, Sakata K, Komatsu S (2014) Phosphoproteomics reveals the effect of ethylene in soybean root under flooding stress. J Proteome Res 13:5618–5634

    Article  CAS  PubMed  Google Scholar 

  27. Olsen JV, de Godoy LMF, Li G, Macek B, Mortensen P, Pesch R, Makarov A, Lange O, Horning S, Mann M (2005) Parts per million mass accuracy on an orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 4:2010–2021

    Article  CAS  PubMed  Google Scholar 

  28. Brosch M, Yu L, Hubbard T, Choudhary J (2009) Accurate and sensitive peptide identification with Mascot Percolator. J Proteome Res 8:3176–3181

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ishihama Y, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J, Mann M (2005) Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics 4:1265–1272

    Article  CAS  PubMed  Google Scholar 

  30. Briesemeister S, Rahnenführer J, Kohlbacher O (2010) YLoc—an interpretable web server for predicting subcellular localization. Nucleic Acids Res 38:497–502

    Article  Google Scholar 

  31. Horton P, Park KJ, Obayashi T, Fujita N, Harada H, Adams-Collier CJ, Nakai K (2007) WoLF PSORT: protein localization predictor. Nucleic Acids Res 35:W585–W587

    Article  PubMed  PubMed Central  Google Scholar 

  32. Chou KC, Shen HB (2010) Plant-mPLoc: a top-down strategy to augment the power for predicting plant protein subcellular localization. PLoS One 5:e11335

    Article  PubMed  PubMed Central  Google Scholar 

  33. Brameier M, Krings A, MacCallum RM (2007) NucPred-predicting nuclear localization of proteins. Bioinformatics 23:1159–1160

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgment

This work was supported by JSPS KAKENHI Grant Number 15H04445.

Author information

Authors and Affiliations

  1. University of Tsukuba, Tsukuba, 305-8572, Japan

    Setsuko Komatsu

Authors
  1. Setsuko Komatsu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Setsuko Komatsu .

Editor information

Editors and Affiliations

  1. Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany

    Hans-Peter Mock

  2. Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany

    Andrea Matros

  3. Leibniz Institute of Vegetable and Ornamental Crops, Großbeeren, Germany

    Katja Witzel

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Komatsu, S. (2018). Isolation, Purity Assessment, and Proteomic Analysis of Nuclei. In: Mock, HP., Matros, A., Witzel, K. (eds) Plant Membrane Proteomics. Methods in Molecular Biology, vol 1696. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7411-5_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7411-5_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7409-2

  • Online ISBN: 978-1-4939-7411-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation