ProteoExtract^® Subcellular Proteome Extraction Kit

sufficient for 20 extractions

Calbiochem^®

OK to freeze
avoid repeated freeze/thaw cycles

sample type: mammalian cultured cells
sample type: mammalian tissue(s)

ambient

2-8℃

1 kit in Glass bottle

Fast and reproducible extraction of subcellular proteomes from mammalian cells

ProteoExtract<TMSYMBOL></TMSYMBOL> 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.

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.

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

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.

Appropriate samples types include:

• Adherent tissue culture cells
• Suspension-grown tissue culture cells
• Frozen cell pellets
• Fragmented tissue

Kit 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. <div class="Bio_doc_image">
Table 2: Buffer Volumes Required for S-PEK Extraction Based on Cell Number
*Tested on rat liver and bovine liver tissue.
</div>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.<div class="Bio_doc_image">Table 3: Buffer Volumes Required for S-PEK Extraction Based on Flask/Dish Size
</div>

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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

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

Upon arrival store the components of the kit under the following conditions:
<div class="Bio_doc_image">Table 1: Storage Information
</div>
*Prior to performing the extraction protocol all frozen buffers must thawed at room temperature. A water bath set at 25℃ will aid in the thawing process. After thawing, mix the buffers well gentle shaking or vortexing.

10 - Combustible liquids