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539790 ProteoExtract® Subcellular Proteome Extraction Kit

ProteoExtract® Subcellular Proteome Extraction Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

Synonyms: S-PEK Kit

539790
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Overview

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Catalogue NumberPackaging Qty/Pack
539790-1KIT Sklená flaša 1 kit
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Description
Overview

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.
Catalogue Number539790
Brand Family Calbiochem®
SynonymsS-PEK Kit
Features and benefits
  • Stepwise extraction resulting in four distinct subcellular proteomes from one sample
  • Highly reproducible
  • No ultracentrifugation steps
  • Fast—needs just 2 hours with 45 minutes hands-on time
  • Produces proteins suitable for functional studies
Application Data
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.

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.
Materials Required but Not Delivered• Platform mixer e.g. IKA Vibramax (when extracting adherent tissue culture cells)
• Cell culture equipment, medium, etc. for cell growth (e. g., RPMI, DMEM)
• Micropipettes and tips, 10 µl, 200 µl and 1 ml size (e.g., Eppendorf, Gilson or equivalent)
• Cooled centrifuge and rotor for 50 ml tube size (Eppendorf, Heraeus, Nalgen, etc.)
• Cooled micro centrifuge and rotor up to 10,000 x g for 2 ml tube size (e.g. Eppendorf)
• Thermo mixer or rolling facility (e.g., Eppendorf)
• Rotary shake (when extracting suspension cells, frozen pellets, and fragmented tissue)
• ProteoExtract® Tissue Dissociation Buffer Kit (Cat. No. 539720) (optional for preparation of cells from fragmented tissue)
• ProteoExtract® Protein Precipitation Kit (Cat. No. 539180; optional for precipitation of proteins from fractions for downstream applications as needed)
References
ReferencesZhang, 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
Product Information
Form20 Extractions
Kit containsWash 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.
Applications
Application ReferencesSabio, 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.
Biological Information
Sample TypeMammalian tissue, cultured mammalian cells
Safety Information
R PhraseR: 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.
S PhraseS: 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).
Product Usage Statements
Intended useThe Calbiochem® ProteoExtract® Subcellular Proteome Extraction Kit is designed for the subcellular extraction of mammalian proteins from the cytosolic, organelle and membrane, nuclear, and cytoskeletal fractions of adherent tissue culture cells, suspension tissue culture cells, frozen cell pellets, and fragmented tissues.
Storage and Shipping Information
Ship Code Ambient Temperature Only
Toxicity Multiple Toxicity Values, refer to MSDS
Storage +2°C to +8°C
Storage ConditionsUpon arrival store the components of the kit under the following conditions:

Table 1: Storage Information


*Prior to performing the extraction protocol all frozen buffers must thawed at room temperature. A water bath set at 25°C will aid in the thawing process. After thawing, mix the buffers well gentle shaking or vortexing.
Avoid freeze/thaw Avoid freeze/thaw
Do not freeze Ok to freeze
Supplemental Information
Kit containsWash 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.
Global Trade Item Number
Catalogue Number GTIN
539790-1KIT 04055977269253