General description

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.

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

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.

Warning

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

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.

Preparation Note

Kit Components Needed for One Extraction

The 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

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.

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-Verlag
Mejdoubi, 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

Legal Information

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

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

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

usage: sufficient for 20 . extractions. manufacturer/tradename: Calbiochem^®.. storage condition: OK to freeze, avoid repeated freeze/thaw cycles. input: sample type: mammalian tissue(s)
sample type: mammalian cultured cells. shipped in: ambient. storage temp.: 2-8Ã‚Â°C. Storage Class Code: 10 - Combustible liquids.