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 | 2 to 8°C |  | Ambient |  | Multiple Toxicity Values, refer to MSDS | | Note: Store and Ship conditions may differ. | | See Key |
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| | ProteoExtract® Subcellular Proteome Extraction Kit |
|  | Cat. No. 539790 |
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S-PEK Kit | Form: 20 Extractions | | Sample Type: Mammalian tissue, cultured mammalian cells | | Kit Contents: Wash buffer, Extraction Buffer I, Extraction Buffer II, Extraction Buffer III, Extraction Buffer IV, Protease Inhibitor Cocktail, Benzonase® Nuclease (Cat. No. 71206), and a user protocol. | | Comments: Fast and reproducible extraction of subcellular proteomes from mammalian cells ProteoExtract® Subcellular Proteome Extraction Kit (S-PEK) is designed for fast and reproducible extraction of subcellular proteomes from adherent and suspension-grown mammalian cells. The S-PEK takes advantage of the different solubilities of certain subcellular compartments in the four selected reagents. In the case of adherent cells, the procedure is performed directly in the tissue culture dish without the need for cell removal. Cells or the parts of the cells remain attached to the plate during sequential extraction of subcellular compartments, until the appropriate extraction reagent is used. Thus, the early destruction of the cellular structure by enzymatic or mechanical detachment of cells from the tissue culture plate and any mixing of different subcellular compartments is prevented. For suspension-grown cells, extraction starts with gentle sedimentation and washing of the cells. The stepwise extraction delivers four distinct protein fractions from one sample: - Cytosolic fraction (F1)
- Membrane/organelle protein fraction (F2)
- Nucleic protein fraction (F3)
- Cytoskeletal fraction (F4)
Proteins are obtained in the native state making the S-PEK suitable for many downstream applications such as 1D and 2D gel electrophoresis, immunoblotting, enzyme activity assays, and protein microarrays. Sample size: 3-5x106 or 25-50 mg tissue. | | Ref.: Zhang, L., and Insel, P. A. 2004. J. Biol. Chem. 279, 20858. Yuan, X., et al. 2002. Electrophoresis 23, 1185. Butcher, et al. 2001. J. Immunol. 167, 2193. Ott, et al. 2001. Pharmacogenomics J. 1, 142. Allen, L. 2000. Nature 405, 819. Dunn, M. J. 2000. Electrophoresis 6. Rabilloud, T. 2000. Two-dimensional Gel Electrophoresis and Identification Methods Springer-Verlag Mejdoubi, et al. 1999. Biochem. Biophys. Res. Comm. 254, 93. Reymond, et al. 1997. Electrophoresis 18, 2842. Laemmli, U. K. 1970. Nature 227, 680. Lowry, et al. 1951. J. Biol. Chem. 193, 265. http://www.expasy.ch/ and http://www.expasy.proteome.org.au Application References Sabio, G., et al. 2005. EMBO J. in press. Efanov, A.M., et al. 2004. Diabetes 53, s75. Singh, L.P., et al. 2004. Am. J. Physiol. Renal Physiol. 286, F409. | | R: 22-36/38-52/53; S: 22-26-28.2-36/37-45 |
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Analysis of Protein Distribution Profiles: Translocation of NF-kB. A431 cells were stimulated with 0.2 µg/ml TNF-a for the indicated times. At the end of each induction period the cells were extracted as outlined in the Detailed Protocol for Subcellular Extraction of Proteins from Adherent Cells. The proteins from an aliquot of each fraction were separated by SDS-PAGE and transferred to PVD membrane for Western blot analysis using an antibody specific for NF-kB. The data indicate that there is measurable translocation of NF-kB from the cytoplasm to the nucleas as early as 5 min after TNF-a stimulation.
Buffer Volumes Required for S-PEK Extraction Based on Cell Number. *Tested on rat liver and bovine liver tissue.
Buffer Volumes Required for S-PEK Extraction Based on Flask/Dish Size.
Protein Concentrations of Each Fraction Obtained From Various Cells Following S-.
Protocol Summary for Adherent Tissue Culture Cells.
Protocol Summary for Fragmented Tissue and Frozen Cell Pellets.
Protocol Summary for Suspension-Grown Tissue Culture Cells.
Schematic Representation of S-PEK Extraction From Adherent Cells. A: Adherent SAOS cells were extracted according to the Detailed Protocol for Subcellular Extraction of Proteins From Adherent Cells as outlined above. Images i-iv show the morphology of the cells before and after each extraction step (200-fold enlarged). The SAOS cells remained attached throughout the extraction procedure. B: An aliquot of each fraction from A were subjected to SDS-PAGE analysis (F1-F4 = fractions 1-4). The data demonstrate clear differences in the protein banding patterns among the 4 fractions. C: Aliquots of each fraction from A were separated by SDS-PAGE and transferred to PVD membrane for blotting with the indicated antibodies. For c-Fos, the fractions were subjected to immunoprecipitation, prior to Western blotting, to enrich for any c-Fos present in each fraction. The data demonstrate that each marker protein is specifically enriched within the appropriate fraction.
Storage Information.
Technical Appendix.
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| - John P. Alao, et al. (2006) The cyclin D1 proto-oncogene is sequestered in the cytoplasm of mammalian cancer cell lines. Molecular Cancer 5, 7-17.
- Stephanie Arndt, et al. (2005) Cloning and functional characterization of new Ski homolog, Fussel-18, specifically expressed in neuronal tissues. Laboratory Investigation 85, 1330-1341.
- J Lypowy, IY Chen and M Abdellatif. (2005) An alliance between RAS GTPASE-activating protein, filamin C, and G3BP regulates myocyte growth.. Journal of Biological Chemistry In Press,.
- M Mourtada-Maarabouni, et al. (2005) Functional expression cloning reveals a central role for the receptor for activated protein kinase C 1 (RACK1) in T cell apoptosis.. Journal of Leukocyte Biology In Press,.
- S Saika S, et al. (2005) Expression of Smad7 in mouse eyes accelerates healing of corneal tissue after exposure to alkali.. American Journal of Pathology 166, 1405-1418.
- Guadalupe Sabio, et al. (2005) p38γ regulates the localisation of SAP97 in the cytoskeleton by modulating its interaction with GKAP. The EMBO Journal 24, 1134-1145.
- Y.S. Song, Y.S. Lee and P.H. Chan. (2005) Oxidative stress transiently decreases the IKK complex (IKKα, β, and γ), an upstream component of NF-κB signaling, after transient focal cerebral ischemia in mice.. Journal of Cerebral Blood Flow & Metabolism 25, 1301-1311.
- Afsaneh Abdolzade-Bavil, et al. (2004) Convenient and versatile subcellular extraction procedure, that facilitates classical protein expression profiling and functional protein analysis. Proteomics 4, 1397-1405.
- Alexander M. Efanov, et al. (2004) Liver X Receptor Activation Stimulates Insulin Secretion via Modulation of Glucose and Lipid Metabolism in Pancreatic β-Cells. Diabetes 53, S75-S78.
- Alexander M. Efanov, et al. (2004) Liver X receptor activation stimulates insulin secretion via modulation of glucose and lipid metabolism in pancreatic β-cells. Diabetes 53, S75-S78.
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