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539790 ProteoExtract® Subcellular Proteome Extraction Kit

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Synonymes: S-PEK Kit

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539790
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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
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      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
      Packaging Information
      Transport Information
      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.
      Specifications
      Global Trade Item Number
      Référence GTIN
      539790-1KIT 04055977269253

      Documentation

      Required Licenses

      Title
      PRODUCTO REGULADO POR LA SECRETARÍA DE SALUD

      ProteoExtract® Subcellular Proteome Extraction Kit FDS

      Titre

      Fiche de données de sécurité des matériaux (FDS) 

      ProteoExtract® Subcellular Proteome Extraction Kit Certificats d'analyse

      TitreNuméro de lot
      539790

      Références bibliographiques

      Aperçu de la référence bibliographique
      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

      Brochure

      Titre
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      ProteoExtract Kits Brochure
      Western Blotting Tools

      Informations techniques

      Titre
      Protein Blotting Handbook: 6th Edition

      Citations

      Titre
    • 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.
    • Tetsuya Mizutani, et al. (2004) Tyrosine dephosphorylation of STAT3 in SARS coronavirus-infected Vero E6 cells. FEBS Letters 577, 187-192.
    • Karen E. Porter, et al. (2004) Simvastatin reduces human atrial myofibroblast proliferation independently of cholesterol lowering via inhibition of RhoA. Cardiovascular Research 61, 745-755.
    • Karen E. Porter, et al. (2004) Simvastin reduces human atrial myofibroblast proliferation independently of chlosterol lowering via inhibition of RhoA. Cardiovascular Research 61, 745-755.
    • Jonathan Rios-Doria, et al. (2004) Cleavage of b-catenin by calpain in prostate and mammary tumor cells. Cancer Research 64, 7237-7240.
    • J Rios-Doria, et al. (2004) Cleavage of beta-catenin by calpain in prostate and mammary tumor cells.. Cancer Research 64, 7237-7240.
    • Jonathan Rios-Doria, et al. (2004) Cleavage of ß-catenin by calpain in prostate and mammary tumor cells. Cancer Research 64, 7237-7240.
      		•
      Protocole Utilisateur

      Revision23-August-2016 RB
      SynonymsS-PEK Kit
      Form20 Extractions
      StorageUpon 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.
      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.
      BackgroundOne major challenge in functional proteomics is the separation of complex protein mixtures to allow detection of low abundance proteins and provide for quantitative and qualitative analysis of proteins impacted by environmental parameters. Prerequisites for the success of such analysis include standardized and reproducible procedures for sample preparation prior to 1- or 2-DGE and/or liquid chromatography (LC). Due to the complexity of total proteomes and the divergence of protein properties, it is necessary to prepare standardized partial proteomes of a given cell or tissue in order to maximize the coverage of the proteome and to increase the chance to visualizing low-abundance proteins. The unique ProteoExtract® Subcellular Proteome Extraction Kit (S-PEK) enables the differential extraction of proteins according to their subcellular localization. The specialized, mild S-PEK procedure yields the majority of proteins in their native state making it highly suitable for demanding proteomics applications, including enzyme activity assays (e.g. reporter gene assays) and subcellular redistribution assays to monitor protein shuttling (e.g. signaling proteins).
      Principles of the assayThe Calbiochem® ProteoExtract® Subcellular Proteome Extraction Kit contains optimized protocols for extraction of proteins, by subcellular location, from adherent and suspension tissue culture cells, frozen cell pellets, and fragmented tissue. The extraction procedure utilizes 4 extraction buffers, prepared with ultra-pure chemicals, to ensure reproducibility; protease inhibitor cocktail to prevent protein degradation during the extraction procedure; and Benzonase® nuclease to degrade contaminating nucleic acids. S-PEK takes advantage of the differential solubility of proteins in different subcellular compartments and utilizes highly specialized extraction buffers to target specific subcellular compartments and simultaneously preserve the structural integrity of the proteins before and during each sequential extraction.

      For suspension-grown tissue culture cells, the extraction starts with gentle sedimentation and washing of the cells. For frozen cell pellets and fragmented tissue, the extraction starts immediately with resuspension in the first extraction buffer. For adherent cells, the extraction is performed directly in the tissue culture dish; the cells or the parts of the cells remain attached to the plate during the entire extraction procedure until the next extraction buffer is added. As a result, 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. A schematic overview of the S-PEK extraction procedure applied to adherent tissue culture cells is shown in figure 1A, noting the morphological changes of the cells.

      The ProteoExtract® Subcellular Proteome Extraction Kit procedure yields the total proteome fractionated into four sub-proteomes of decreasing complexity. Using Extraction Buffer I, the cytosolic proteins are released (fraction 1). In the second step the membrane and organelle proteins are solubilized using Extraction Buffer II. Extraction Buffer III yields the nuclear proteins (fraction 3). In the final step, using Extraction Buffer IV, the components of the cytoskeleton are solubilized (fraction 4).










      Figure 1: 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.


      The efficiency of the subcellular fractionation by S-PEK has been shown by 1-DGE (Figure 1B) and immunoblotting of selected marker proteins (Figure 1C). More than 90% of subcellular marker proteins can be assigned to the expected subcellular fraction (Figure 1C). Please note that HSP70 is present in both the cytoplasm and the mitochondria of cells and is thus detected in both the cytoplasmic and membrane/organelle fractions using the S-PEK procedure.
      Materials provided• Wash Buffer (Kit Component No. KP31250): 1 bottle, 100 ml
      • Extraction Buffer I (Kit Component No. KP31311): 1 bottle, 22 ml
      • Extraction Buffer II (Kit Component No. KP31312): 1 bottle, 22 ml
      • Extraction Buffer III (Kit Component No. KP31313): 1 bottle, 11 ml
      • Extraction Buffer IV (Kit Component No. KP31314): 1 bottle, 10 ml
      • Protease Inhibitor Cocktail (Kit Component No. KP31288): 1 vial, 450 µl
      • Benzonase®(≥250 U/µl) (Kit Component No. KP31255): 1 vial, 45 µl
      Materials Required but not provided• 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)
      PreparationAppropriate samples types include: • Adherent tissue culture cells • Suspension-grown tissue culture cells • Frozen cell pellets • Fragmented tissue
      Reagent preparationKit Components Needed for One Extraction The volume of each component required for one subcellular extraction depends on the amount of starting material and is shown in the table below.

      Table 2: Buffer Volumes Required for S-PEK Extraction Based on Cell Number

      *Tested on rat liver and bovine liver tissue.

      The following table lists the amount of extraction buffer needed for extracting proteins from cells grown in various size tissue culture flasks and dishes. As guideline to estimate the amount of starting material please refer to the table above for a listing of expected protein yields obtained from selected cell types following S-PEK extraction.

      Table 3: Buffer Volumes Required for S-PEK Extraction Based on Flask/Dish Size
      Detailed protocolProtocol for Subcellular Extraction of Proteins From Adherent Tissue Culture Cells

      This protocol is scaled for the extraction of proteins from cell monolayers in T25 tissue culture flasks (3-5 x 106 cells). Please refer to the tables in the Reagent Preparation Section above to determine the amount of each Extraction Buffer needed for the number of cells or type of tissue culture flask/dish used.

      For optimal adherence of the cells during the extraction procedure, it is vital that the cells are in the logarithmic phase of growth at ~80% confluency. Despite these guidelines, certain cell types are more prone to detachment from the dish during the extraction. If the cells detach at any step during the extraction, proceed with the Protocol for Subcellular Extraction of Proteins from Suspension-Grown Tissue Culture Cells.

      To monitor the extraction phase contrast microscopy can be used to examine morphological changes in the cells (see Figure 1A).

      1. Before beginning the extraction, mix the Wash Buffer and all Extraction Buffers well by vortexing. During the extraction keep Extraction Buffers I-III and Benzonase® nuclease on ice and Extraction Buffer IV and Protease Inhibitor Cocktail at room temperature. The buffers must be completely thawed before starting the extraction.
      2. Carefully remove the cell culture medium without disturbing the cell monolayer.
      3. Wash the cells by carefully overlaying the cell monolayer with 2 ml ice-cold Wash Buffer. Gently agitate the cell culture flask/dish at 4°C for 5 min (Note: if the cells detach, transfer the cells in the Wash Buffer to an appropriate centrifuge tube).
      4. Aspirate the Wash Buffer completely without disturbing the cell monolayer.
      5. Repeat steps 3 and 4 to ensure complete removal of contaminating medium. (Note: if the cells detach, transfer the cells in the Wash Buffer to an appropriate centrifuge tube).
      6. Mix 1 ml ice-cold Extraction Buffer I with 5 µl Protease Inhibitor Cocktail and immediately add the mixture to the flask without disturbing the cell monolayer. Carefully rotate the flask/dish until all cells are covered. Incubate for 10 min at 4°C under gentle agitation (Note: if the cells detach, transfer the cells in the Wash Buffer to an appropriate centrifuge tube).
      7. Using a pipette, transfer the supernatant (fraction 1) to a clean tube; take care not to disturb the remaining cell monolayer. Make sure that all liquid is removed. Store fraction 1 on ice.
      8. Mix 1 ml ice-cold Extraction Buffer II with 5 µl Protease Inhibitor Cocktail and immediately add the mixture to the flask/dish without disturbing the cell monolayer. Carefully rotate the flask/dish until all cells are covered. Incubate for 30 min at 4°C under gentle agitation (Note: if the cells detach, transfer the cells in the Wash Buffer to an appropriate centrifuge tube).
      9. Using a pipette, transfer the supernatant (fraction 2) to a clean tube; take care not to disturb the remaining cell monolayer. Make sure that all liquid is removed. Store fraction 2 on ice.
      10. Mix 500 µl ice-cold Extraction Buffer III with 5 µl Protease Inhibitor Cocktail and 1.5 µl (≥ 375 U) Benzonase® nuclease and immediately add the mixture to the flask/dish without disturbing the cell monolayer. Carefully rotate the flask/dish until all cells are covered. Incubate for 10 min at 4°C under gentle agitation (Note: if the cells detach, transfer the cells in the Wash Buffer to an appropriate centrifuge tube).
      11. Using a pipette, transfer the supernatant (fraction 3) to a clean tube; take care not to disturb the remaining cell monolayer. Completely remove all the liquid. Store fraction 3 on ice.
      12. Mix 500 µl room temperature Extraction Buffer IV with 5 µl Protease Inhibitor Cocktail and immediately add the mixture to the flask/dish. Carefully rotate the flask/dish until all remaining cell components are covered. The remaining cell structures will detach upon treatment with Extraction Buffer IV. After complete solubilization of the residual materials, transfer the extract (fraction 4) to a clean tube. Store fraction 4 on ice. (Please refer to the Technical Appendix, point 4).
      13. If the fractions are to be used immediately, continue to store on ice prior to performing any downstream applications and analyses. For long-term storage, dispense each fraction into aliquots (e.g., 100 µl) and freeze at -20°C or -70°C until use.
      14. For use of the fractions for 1- or 2-DGE refer to the Technical Appendix.

      Protocol for Subcellular Extraction of Proteins From Suspension-Grown Tissue Culture Cells

      This protocol is scaled for extracting proteins from 3-5 x 106 cells. Please refer to the tables in the Reagent Preparation section to determine the volume of Extraction Buffers needed for the number of cells used.

      1. Before beginning the extraction, mix the Wash Buffer and all Extraction Buffers well by vortexing. During the extraction keep Extraction Buffers I-III and Benzonase® nuclease on ice and Extraction Buffer IV and Protease Inhibitor Cocktail at room temperature. The buffers must be completely thawed before starting the extraction.
      2. Transfer the cells to an appropriate centrifuge tube and pellet by centrifugation for 10 min at 100-300 g, 4°C. Aspirate and discard the supernatant without disturbing the cell pellet.
      3. Add 2 ml ice-cold Wash Buffer and resuspend the cell pellet by gently flicking the tube. Incubate for 5 min at 4°C under gentle agitation. The use of a rotary shaker is recommended to avoid formation of cell clumps.
      4. Pellet the cells by centrifugation for 10 min. at 100-300 g, 4°C. Carefully remove and discard the supernatant without disturbing the cell pellet.
      5. Repeat steps 3 and 4 to ensure complete removal of contaminating medium.
      6. Mix 1 ml ice-cold Extraction Buffer I with 5 µl Protease Inhibitor Cocktail and immediately add the mixture to the cell pellet. Resuspend the cell pellet by gently flicking the tube. Incubate for 10 min at 4°C under gentle agitation. The use of a rotary shaker is recommended to avoid formation of cell clumps.
      7. Pellet the insoluble material by centrifugation for 10 min at 500-1000 g, 4°C.
      8. Using a pipette, transfer the supernatant (fraction 1) to a clean tube. Store fraction 1 on ice.
      9. Mix 1 ml ice-cold Extraction Buffer II with 5 µl Protease Inhibitor Cocktail and immediately add the mixture to the cell pellet. Resuspend the cell pellet by gently flicking the tube. Incubate for 30 min at 4°C under gentle agitation. the use of a rotary shaker is recommended to avoid formation of cell clumps.
      10. Pellet the insoluble material by centrifugation for 10 min at 5000-6000 g, 4°C.
      11. Using a pipette, transfer the supernatant (fraction 2) to a clean tube. Store fraction 2 on ice.
      12. Mix 500 µl Extraction buffer III with 5 µl Inhibitor Cocktail and 1.5 µl (≥ 375 U) Benzonase® nuclease and immediately add the mixture to the cell pellet. Resuspend the cell pellet by pipetting up and down. Incubate for 10 min at 4°C under gentle agitation. The use of a rotary shaker is recommended to avoid formation of cell clumps.
      13. Pellet the insoluble material by centrifugation for 10 min at 6800 g, 4°C.
      14. Using a pipette, transfer the supernatant (fraction 3) to a clean tube. Store fraction 3 on ice.
      15. Mix 500 µl room temperature Extraction Buffer IV with 5 µl Protease Inhibitor Cocktail and immediately add the mixture to the cell pellet. Carefully suspend residual particles by pipetting up and down (fraction 4).
      16. If the fractions are to be used immediately, continue to store on ice prior to performing any downstream applications and analyses. For long-term storage, dispense each fraction into aliquots (e.g., 100 µl) and freeze at -20°C or -70°C until use.
      17. For use of the fractions for 1- or 2-DGE refer to the Technical Appendix.

      Protocol for Subcellular Extraction of Proteins From Fragmented Tissue and Frozen Cell Pellets

      This protocol applies to 25-50 mg fresh and flash-frozen fragmented tissue or 3-5 x 106 flash-frozen cells. For preparation of fragmented tissue we recommend the ProteoExtract® Tissue Dissociation Buffer Kit (Cat. No. 539720) or published methods (e.g., Reymond, et. al.

      Important Note: When preparing frozen cell pellets be sure that the cells are washed with a suitable buffer before freezing. Use only flash-frozen (liquid nitrogen) cell pellets or tissues for the extraction.

      1. Mix 1 ml ice-cold Extraction Buffer I with 5 µl Protease Inhibitor Cocktail and immediately add the mixture to the fragmented tissue or frozen cell pellet. Gently resuspend the fragmented tissue or frozen cell pellet by gently flicking the tube. Incubate for 10 min at 4°C under gentle agitation. The use of a rotary shaker is recommended to avoid formation of cell clumps.
      2. Pellet the insoluble material by centrifugation for 10 min at 500-1000 g, 4°C.
      3. Carefully transfer the supernatant (fraction 1) to a clean tube. Store fraction 1 on ice.
      4. Mix 1 ml ice-cold Extraction Buffer II with 5 µl Protease Inhibitor Cocktail and immediately add the mixture to the pellet. Resuspend the pellet by gently flicking the tube. Incubate for 30 min at 4°C under gentle agitation. The use of a rotary shaker is recommended to avoid formation of cell clumps.
      5. Pellet the insoluble material by centrifugation for 10 min at 5000-6000 g, 4°C.
      6. Carefully transfer the supernatant (fraction 2) to a clean tube. Store fraction 2 on ice.
      7. Mix 500 µl Extraction Buffer III with 5 µl Inhibitor Cocktail and 1.5 µl (≥ 375 U) Benzonase® nuclease and immediately add the mixture to the pellet. Resuspend the pellet by gently flicking the tube. Incubate for 10 min at 4°C under gentle agitation. The use of a rotary shaker is recommended to avoid formation of cell clumps.
      8. Pellet the insoluble material by centrifugation for 10 min at 7000 g, 4°C.
      9. Carefully transfer the supernatant (fraction 3) to a clean tube. Store fraction 3 on ice.
      10. Mix 500 µl room temperature Extraction Buffer IV with 5 µl Protease Inhibitor Cocktail and immediately add the mixture to the pellet. Resuspend the residual particles by pipetting up and down (fraction 4).
      11. If the fractions are to be used immediately, continue to store on ice prior to performing any downstream applications and analyses. For long-term storage, dispense each fraction into aliquots (e.g., 100 µl) and freeze at -20°C or -70°C until use.
      12. For use of the fractions for 1- or 2-DGE refer to the Technical Appendix.
      Example dataS-PEK has been used successfully with a wide variety of human tissue culture cells. The cells were grown in T25 tissue culture flasks with approximately 1 x 10⁶ cells/flask at approximately 80% confluence. Please note that the actual number of cells at 80% confluency may change considerably among different cell types. The table below lists the quantity of protein obtained in each fraction following S-PEK extraction.

      Table 4: Protein Concentrations of Each Fraction Obtained From Various Cells Following S-

      The S-PEK procedure is ideally suited for investigating changes in subcellular localization of regulatory proteins under certain experimental conditions or in certain disease states. To demonstrate this application, the translocation of NFκB from the cytosol to the nucleus upon stimulation of cells with tumor necrosis factor α (TNFα) (Mejdoubi et al., 1999; Butcher et al., 2001) was chosen as a model system. NFκB translocation was studied in TNFα-stimulated A431 cells that were subsequently extracted with the S-PEK kit. Nuclear translocation of NFκB is easily demonstrated by immunoblotting of the 4 fractions and by densitometric analysis of filters (see figure below). The control protein, calpain, did not undergo any translocation between fractions following TNFα-stimulation of cells (data not shown). Thus, using the S-PEK procedure, translocation of regulatory proteins can be investigated.




      Figure 2: Analysis of Protein Distribution Profiles: Translocation of NF-κB

      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.
      Technical AppendixProblem Upon centrifugation of suspension-grown cells, no compact pellet is formed. Answer It cannot be ruled out that certain types of cells do not form compact pellets using an acceleration of 100 g. In these cases increase the acceleration of the centrifuge to approximately 300 g, for example.
      Problem Upon washing or extraction of adherent tissue culture cells, detachment of the cell monolayer occurs. Answer When adherent cell detach during the extraction steps transfer the resulting cell suspension to a clean tube and continue with the extraction respective step using the Detailed Protocol for Subcellular Extraction of Proteins from Suspension-Grown Cells. This does normally not affect the quality of the results.
      Problem How do I determine the protein concentration of the subcellular protein extracts? Answer As the extraction buffers contain components that might interfere with protein quantification assays specific protein assays such as the RcDc-Kit (BioRad) or the Non-interfering™ Protein Assay Kit (Cat. No. 488250) are required to determine the protein concentration. Alternatively you may precipitate an aliquot of the extract prior quantification using the ProteoExtract® Protein Precipitation Kit (Cat. No. 539180).
      Problem When storing Extraction Buffer 4 or fraction 4 on ice, a precipitate occurs. Answer This does not normally affect the results of the extraction or downstream experiments. If a precipitate forms in Extraction Buffer 4 or fraction 4, gently warm the sample to room temperature, mix well, and use immediately for extraction or analysis, respectively.
      Problem How do I prepare subcellular fractions generated with S-PEK for one-dimensional SDS-PAGE? Answer S-PEK fractions can be directly analyzed by one-dimensional SDS-PAGE: Dilute the sample with an equal volume of 2X SDS-PAGE sample buffer (e.g., 125 mM Tris-HCl, pH 6.8; 10% (w/v) SDS; 30% (v/v) glycerol; 100 mM DTT; 0.002% (w/v) bromophenol blue) and heat to 95°C for 5 min prior to loading the gel.
      Problem How can I concentrate S-PEK fractions? Answer If the protein concentration of fractions is not sufficient for your downstream applications, we recommend using the ProteoExtract® Protein Precipitation Kit (Cat. No. 539180) to concentrate the proteins. The resulting protein pellet can be resuspended in a buffer suitable for your downstream applications.
      Problem How do I prepare S-PEK fractions for two-dimensional SDS-PAGE? Answer When the protein concentrations are high enough, S-PEK fractions 1, 2, and 3 can be used directly for 2-DGE. Samples must be diluted 1: 4 with a common loading buffer for IEF (e.g., 5 M urea, 2 M thiourea, 4% CHAPS, ampholytes, 100 mM DTT) and incubated for 60 min at room temperature prior to loading on an IEF gel. Fraction 4 must include a clean-up step (e.g, using the ProteoExtract® Protein Precipitation Kit as outlined above), prior to loading the proteins on an IEF gel. For improved results in 2-DGE we strongly recommend precipitation (clean-up) of all 4 fractions prior to IEF.















      Protocol Summary

      Figure 3: Protocol Summary for Adherent Tissue Culture Cells


      Figure 4: Protocol Summary for Suspension-Grown Tissue Culture Cells


      Figure 5: Protocol Summary for Fragmented Tissue and Frozen Cell Pellets
      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.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Biosciences, inc.
      Benzonase® and ProteoExtract® are registered trademarks of Merck KGaA, Darmstadt, Germany.