Skip to main content

Isolation of Cytoplasmatic Proteins from Cultured Cells for Two-Dimensional Gel Electrophoresis

  • Protocol
2D PAGE: Sample Preparation and Fractionation

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 425))

Summary

Cytoplasma is the cell interior place between the cellular membrane and the nucleus, where various intracellular activities take place, including energy production, reactive oxygen species (ROS) detoxification, heme synthesis, nitrogen and lipid metabolism, phosphorylation in signal transduction, and cytoskeletal meshwork construction. The rich cytoplasmatic proteins carrying out these intracellular functions are interesting targets for biochemical and molecular biological studies. The relatively recent discipline of proteomics offers a chance to globally analyze the changes in cytoplasmic proteins corresponding to drug treatments or disease conditions, and thus provide target candidates for further biological validation in drug development and biomarker discovery. Isolation of cytoplasmic proteins from cells is a necessary step for high resolution protein separation by two-dimensional gel electrophoresis (2DE) and specific proteomic analysis.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
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 109.99
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. Mesecke N., Terziyska N., Kozany C., Baumann F., Neupert W., Hell K., Herrmann J.M. (2005) A disulfide relay system in the intermembrane space of mitochondria that mediates protein import, Cell 121, 1059–69.

    Article  CAS  PubMed  Google Scholar 

  2. Ferret P. J., Hammoud R., Tulliez M., etal. (2001) Detoxification of reactive oxygen species by a nonpeptidyl mimic of superoxide dismutase cures acetaminophen-induced acute liver failure in the mouse, Hepatology 33, 1173–80.

    Article  CAS  PubMed  Google Scholar 

  3. Szaszi K., Jones J. J., Nathens A. B., etal. (2005) Glutathione depletion inhibits lipopolysaccharide-induced intercellular adhesion molecule 1 synthesis, Free Radic.Biol.Med. 38, 1333–43.

    Article  CAS  PubMed  Google Scholar 

  4. Zou M. H., Kirkpatrick S. S., Davis B. J., etal. (2004) Activation of the AMP-activated protein kinase by the anti-diabetic drug metformin in vivo. Role of mitochondrial reactive nitrogen species, J.Biol.Chem. 279, 43940–51.

    Article  CAS  PubMed  Google Scholar 

  5. Osawa Y.,Uchinami H., Bielawski J., Schwabe R. F.,Hannun Y. A., Brenner D.A. (2005) Roles for C16-ceramide and sphingosine 1-phosphate in regulating hepatocyte apoptosis in response to tumor necrosis factor-alpha, J.Biol.Chem. 280, 27879–87.

    Article  CAS  PubMed  Google Scholar 

  6. Nakamura Y., Awai K., Masuda T., Yoshioka Y., Takamiya K., Ohta H. (2005) A novel phosphatidylcholine-hydrolyzing phospholipase C induced by phosphate starvation in Arabidopsis, J.Biol.Chem. 280, 7469–76.

    Article  CAS  PubMed  Google Scholar 

  7. Favoreel H. W., Van Minnebruggen G., Adriaensen D.,Nauwynck H. J. 2005) Cytoskeletal rearrangements and cell extensions induced by the US3 kinase of an alphaherpesvirus are associated with enhanced spread, Proc.Natl.Acad.Sci.U.S.A 102, 8990–5.

    Google Scholar 

  8. Yang T.,Chaudhuri S.,Yang L., Chen Y., Poovaiah B. W. (2004) Calcium/calmodulin up-regulates a cytoplasmic receptor-like kinase in plants, J.Biol.Chem. 279, 42552–559.

    Article  CAS  PubMed  Google Scholar 

  9. Jiang X. S., Dai J., Sheng Q. H., etal. (2005) A comparative proteomic strategy for subcellular proteome research: ICAT approach coupled with bioinformatics prediction to ascertain rat liver mitochondrial proteins and indication of mitochondrial localization for catalase, Mol.Cell Proteomics 4, 12–34.

    CAS  PubMed  Google Scholar 

  10. He Q. Y. and Chiu J. F. (2003) Proteomics in biomarker discovery and drug development, J.Cell Biochem. 89, 868–86.

    Article  CAS  PubMed  Google Scholar 

  11. Wang Y.,Chiu J. F., and He Q. Y. (2005) Proteomics in computer-aided drug design, Current Computer-Aided Drug Design 1, 43–52.

    Article  Google Scholar 

  12. Wang Y., He Q. Y., Sun R. W.,Che C. M., Chiu J. F. (2005) Gold(III) Porphyrin 1a induced apoptosis by mitochondrial death pathways related to reactive oxygen species, Cancer Res. 65, 11553–64.

    Article  CAS  PubMed  Google Scholar 

  13. Bidere N., Lorenzo H. K., Carmona S., Laforge M., Harper F.,Dumont C., Senik A. (2003) Cathepsin D triggers Bax activation, resulting in selective apoptosis-inducing factor (AIF) relocation in T lymphocytes entering the early commitment phase to apoptosis, J.Biol.Chem. 278, 31401–11.

    Article  CAS  PubMed  Google Scholar 

  14. Arnoult D.,Gaume B.,Karbowski M., Sharpe J. C.,Cecconi F.,Youle R. J. (2003) Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization, EMBO J. 22, 4385–99.

    Article  CAS  PubMed  Google Scholar 

  15. Arnoult D.,Parone P.,Martinou J. C., Antonsson B., Estaquier J., Ameisen J. C. (2002) Mitochondrial release of apoptosis-inducing factor occurs downstream of cytochrome c release in response to several proapoptotic stimuli, J.Cell Biol. 159, 923–9.

    Article  CAS  PubMed  Google Scholar 

  16. Sanchez-Alcazar J. A., Bradbury D. A., Pang L.,Knox A. J. (2003) Cyclooxygenase (COX) inhibitors induce apoptosis in non-small cell lung cancer through cyclooxygenase independent pathways, Lung Cancer 40, 33–44.

    Article  PubMed  Google Scholar 

  17. Vahsen N., Cande C.,Briere J. J., etal. (2004) AIF deficiency compromises oxidative phosphorylation, EMBO J. 23, 4679–89.

    Article  CAS  PubMed  Google Scholar 

  18. Wang H., Yu S. W., Koh D. W., etal. (2004) Apoptosis-inducing factor substitutes for caspase executioners in NMDA-triggered excitotoxic neuronal death, J.Neurosci. 24, 10963–73.

    Article  CAS  PubMed  Google Scholar 

  19. Yu S. W., Wang H., Poitras M. F., etal. (2002) Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor, Science 297, 259–63.

    Article  CAS  PubMed  Google Scholar 

  20. Otera H., Ohsakaya S.,Nagaura Z., Ishihara N., Mihara K. (2005) Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space, EMBO J. 24, 1375–86.

    Article  CAS  PubMed  Google Scholar 

  21. Leung Y. K.,Lau K. M.,Mobley J., Jiang Z.,Ho S. M. (2005) Overexpression of cytochrome P450 1A1 and its novel spliced variant in ovarian cancer cells: alternative subcellular enzyme compartmentation may contribute to carcinogenesis, Cancer Res. 65, 3726–34.

    Article  CAS  PubMed  Google Scholar 

  22. Jayanthi S., Deng X.,Noailles P. A., Ladenheim B.,Cadet J. L. (2004) Methamphetamine induces neuronal apoptosis via cross-talks between endoplasmic reticulum and mitochondria-dependent death cascades, FASEB J. 18, 238–51.

    Article  CAS  PubMed  Google Scholar 

  23. Gallego M. A., Joseph B.,Hemstrom T. H., etal. (2004) Apoptosis-inducing factor determines the chemoresistance of non-small-cell lung carcinomas, Oncogene 23, 6282–91.

    Article  CAS  PubMed  Google Scholar 

  24. Richard I.,Broux O., Allamand V., etal. (1995) Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A, Cell 81, 27–40.

    Article  CAS  PubMed  Google Scholar 

  25. Marceau N. and Loranger A. (1995) Cytokeratin expression, fibrillar organization, and subtle function in liver cells, Biochem.Cell Biol. 73, 619–25.

    CAS  PubMed  Google Scholar 

  26. Parry D. A. (1995) Hard alpha-keratin IF: a structural model lacking a head-to-tail molecular overlap but having hybrid features characteristic of both epidermal keratin and vimentin IF, Proteins 22, 267–72.

    Article  CAS  PubMed  Google Scholar 

  27. Watanabe M., Sasaki M., Itoh K., etal. (2005) JunB induced by constitutive CD30-extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase signaling activates the CD30 promoter in anaplastic large cell lymphoma and reed-sternberg cells of Hodgkin lymphoma, Cancer Res. 65, 7628–34.

    CAS  PubMed  Google Scholar 

  28. Moreno-Manzano V., Ishikawa Y.,Lucio-Cazana J., and Kitamura M. (1999) Suppression of apoptosis by all-trans-retinoic acid. Dual intervention in the c-Jun n-terminal kinase-AP-1 pathway, J.Biol.Chem. 274, 20251–58.

    Article  CAS  PubMed  Google Scholar 

  29. Ozaki T., Katsumoto E.,Mui K., Furutsuka D., Yamagami S. (1998) Distribution of Fos- and Jun-related proteins and activator protein-1 composite factors in mouse brain induced by neuroleptics, Neuroscience 84, 1187–96.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This investigation was partially supported by grants from Hong Kong University funding (No. 200511159099 to Q.Y.H.) and the Area of Excellence Scheme of the Hong Kong University Grants Committee.

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2008 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Wang, Y., Chiu, JF., He, QY. (2008). Isolation of Cytoplasmatic Proteins from Cultured Cells for Two-Dimensional Gel Electrophoresis. In: Posch, A. (eds) 2D PAGE: Sample Preparation and Fractionation. Methods in Molecular Biology™, vol 425. Humana Press. https://doi.org/10.1007/978-1-60327-210-0_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-60327-210-0_9

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60327-209-4

  • Online ISBN: 978-1-60327-210-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics