Components

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

Stepwise extraction resulting in four distinct subcellular proteomes from one sample Highly reproducible No ultracentrifugation steps FastR12;needs just 2 hours with 45 minutes hands-on time Produces proteins suitable for functional studies

General description

Fast and reproducible extraction of subcellular proteomes from mammalian cellsProteoExtract^® 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.

Legal Information

Benzonase is a registered trademark of Merck KGaA, Darmstadt, Germany

PROTEOEXTRACT is a registered trademark of Merck KGaA, Darmstadt, Germany

CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany

Other Notes

Zhang, L., and Insel, P. A. 2004. J. Biol. Chem.279, 20858.Yuan, X., et al. 2002. Electrophoresis23, 1185.Butcher, et al. 2001. J. Immunol.167, 2193.Ott, et al. 2001. Pharmacogenomics J.1, 142.Allen, L. 2000. Nature405, 819.Dunn, M. J. 2000. Electrophoresis 6.Rabilloud, T. 2000. Two-dimensional Gel Electrophoresis and Identification Methods Springer-VerlagMejdoubi, et al. 1999. Biochem. Biophys. Res. Comm.254, 93.Reymond, et al. 1997. Electrophoresis18, 2842.Laemmli, U. K. 1970. Nature227, 680.Lowry, et al. 1951. J. Biol. Chem.193, 265.http://www.expasy.ch/ and http://www.expasy.proteome.org.au

Packaging

1 kit in Glass bottle

Preparation Note

Kit Components Needed for One ExtractionThe volume of each component required for one subcellular extraction depends on the amount of starting material.

Appropriate samples types include: Adherent tissue culture cells Suspension-grown tissue culture cells Frozen cell pellets Fragmented tissue

Principle

The 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 Stability

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.

Warning

Toxicity: Multiple Toxicity Values, refer to MSDS (O)