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ProteoExtract® Subcellular Proteome Extraction Kit
 | | | Fast and reproducible extraction of subcellular proteomes from mammalian cells...
 Overview:
ProteoExtract® Subcellular Proteome Extraction Kit (S-PEK) is designed for fast and reproducible extraction of subcellular proteomes from mammalian tissue and adherent and suspension-grown cells.
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. For tissues, fragmentation is required before proceeding with the extraction protocol.
The stepwise extraction delivers four distinct protein fractions from one sample:
• Cytosolic protein fraction
• Membrane/organelle protein fraction
• Nuclear protein fraction
• Cytoskeletal protein fraction
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.
Features and Benefits:
- Stepwise extraction resulting in four distinct subcellular proteomes from one sample
- Highly reproducible
- No ultracentrifugation steps
- Fast— just 2 hours, only 45 minutes hands-on time
- Produces proteins suitable for functional studies | | | Procedure:
The stepwise extraction delivers 4 distinct protein fractions from one sample: Cytosolic fraction (F1), Membrane/organelle protein fraction (F2), Nucleic protein fraction (F3), Cytoskeletal fraction (F4). | | Morphological changes of cells during subcellular extraction. |
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| | | | Applications:
The innovative buffer system used in the S-PEK yields proteins directly suitable for various proteomics downstream applications. Because proteomes are obtained in the native state, proteins are particularly suited for sensitive applications such as enzyme activity assays or microarrays. | | | • Protein Profiling by 1D/2DGE
Subcellular fractionation of cells using the S-PEK procedure can be used as sample preparation for two-dimensional gel electrophoresis (2DGE). A comparative analysis of subcellular proteomes by 2DGE indicated a large number of protein spots to be unique to each of the respective subcellular fractions (see figure below). Protein patterns of membrane/organelle and nucleic fractions are clearly distinct from the protein patterns of cytosolic and cytoskeletal fraction. Thus the subcellular prefractionation can increase the chance to visualize low-abundant proteins in membranes/organelles and nucleus since these are efficiently separated from high-abundant proteins in cytosol and cytoskeleton. | | | | | | • Western Blotting
Stepwise extractions show clearly distinct protein patterns of different subcellular fractions (F1-4 = Fraction 1-4) | | A. | | |  | | |
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| | | • Enzyme Activity Assays
Proteome fractions prepared with the S-PEK may be used for most enzyme assays including reporter gene assays, kinases, and immunoassays. | | | | • Subcellular Redistribution Assays
Innovative extraction buffers in the S-PEK solubilize distinct subcellular compartments. The S-PEK provides a valuable tool for the evaluation and mechanistic studies of protein redistribution events, such as time and dose dependent relocalization of signaling proteins upon stimulation of cells. | | | |
 Add ProteoExtract® Subcellular Proteome Extraction Kit (Cat. No. 539790) to your shopping cart now:
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| * NOTE: In Stock status is based on item availability worldwide. |
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| | Add ProteoExtract® Subcellular Proteome Extraction Kit, Mini (Cat. No. 539791) to your shopping cart now:
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| * NOTE: In Stock status is based on item availability worldwide. |
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| | | | Selected Citation 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. | | Yu, L., et al. 2005 Invest Ophthalmol. Vis. Sci 46, 1726. | | Lypowy, J., et al. 2005. J. Biol. Chem. in press. | | Mourtada-Maarabouni, M., et al. 2005. J. Leukoc. Biol in press. | | Saika, S., et al. 2005. Am. J. Pathol. 166, 1405. | | Rios-Doria, J., et al. 2004. Cancer Research 64, 7237. |
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