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15 February 2012
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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.| Product information | |||
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| Form | 20 Extractions | ||
| Avoid freeze/thaw | Yes | ||
| Sample type | Mammalian tissue, cultured mammalian cells | ||
| Kit contains | 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. | ||
| Features and benefits |
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| Store and ship information | |||
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| Storage | +2°C to +8°C | ||
| Ship |
Ambient Temperature Only
Multiple Toxicity Values, refer to MSDS |
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| Safety information | |||
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| S Phrase |
S: 22-26-28.2-36/37-45 Do not breathe dust. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. After contact with skin, wash immediately with soap and plenty of water. Wear suitable protective clothing and gloves. In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible). |
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| R Phrase |
R: 22-36/38-52/53 Harmful if swallowed. Irritating to eyes and skin. Harmful to aquatic organisms, may cause long-term adverse effects in the aquatic environment. |
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| Data | |||
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 <b>A:</b> 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>B:</b> An aliquot of each fraction from <b>A</b> 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. <b>C:</b> Aliquots of each fraction from <b>A</b> 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.  A431 cells were stimulated with 0.2 µg/ml TNF-α 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-κB. The data indicate that there is measurable translocation of NF-κB from the cytoplasm to the nucleas as early as 5 min after TNF-α stimulation.   *Tested on rat liver and bovine liver tissue.      
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