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.
Key Words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
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.
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.
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.
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.
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.
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.
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.
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.
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.
He Q. Y. and Chiu J. F. (2003) Proteomics in biomarker discovery and drug development, J.Cell Biochem. 89, 868–86.
Wang Y.,Chiu J. F., and He Q. Y. (2005) Proteomics in computer-aided drug design, Current Computer-Aided Drug Design 1, 43–52.
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.
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.
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.
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.
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.
Vahsen N., Cande C.,Briere J. J., etal. (2004) AIF deficiency compromises oxidative phosphorylation, EMBO J. 23, 4679–89.
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.
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.
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.
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.
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.
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.
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.
Marceau N. and Loranger A. (1995) Cytokeratin expression, fibrillar organization, and subtle function in liver cells, Biochem.Cell Biol. 73, 619–25.
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.
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.
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.
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.
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
Editor information
Editors and Affiliations
Rights 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