ProteoExtract^® Subcellular Proteome Extraction Kit, Mini

The ProteoExtract^® Subcellular Proteome Extraction Kit, Mini is a tool for the differential extraction of proteins from mammalian cells according to their subcellular localization. The sequential extraction steps yield proteins in their native state, making it highly suited for demanding proteomics applications. Sample size: 3-5x10⁶ or 25-50 mg tissue.

The Calbiochem^® ProteoExtract Subcellular Proteome Extraction Kit Mini (S-PEK) is intended for the differential extraction of proteins from mammalian cells according to their subcellular localization. The procedure yields proteins in their native state making it highly suited 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).

• Extraction Buffers I-III: Before extraction, buffers must be thawed at room temperature. A water bath at 25°C will speed up the process. After thawing, mix components by gently shaking or vortexing.• Protease Inhibitor Cocktail: During sample preparation it must be kept at room temperature to prevent freezing of DMSO.• Benzonase^® Nuclease: During the extraction keep the enzyme on ice.

The ProteoExtract^® Subcellular Proteome Extraction Kit, Mini is designed for mammalian samples:

• Adherent tissue culture cells
• Suspension grown tissue culture cells
• Frozen cell pellets
• Fragmented tissues S-PEK has been successfully used with a wide variety of human cells.

The cells are grown in T25-culture flasks with about 1 x 10⁶ cells/flask at approximately 80% confluence. Please note that the actual number of cells at 80% confluence of the monolayer may change considerably amongst different cell types. The buffer volumes given in the extraction protocols have been scaled up for the use of T25-culture flasks.<p>Protein concentrations of cellular fractions obtained using S-PEK and adherent tissue culture cells. Protein amounts listed in the table were obtained following extraction of cell monolayers grown to 80% confluency in T25-flasks. The values are provided only as guide for estimating the amount of starting material needed to achieve a certain protein yield using S-PEK extraction. Please note that different tissue culture cells may yield considerably different protein amounts. The values represented in the table are mean values of independent experiments, therefore the total protein content may differ.
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Upon arrival store the components of the kit under the following conditions:



• Wash Buffer: Store at 4°C.

• Extraction Buffers I-III: Store at 4°C for up to 6 months. For long-term storage, freeze the buffers in convenient aliquots at -20°C.

• Extraction Buffer IV: Store at room temperature.

• Protease Inhibitor Cocktail: The cocktail is supplied in DMSO and can be stored at 4°C for up to 6 months. For prolonged storage, freeze the cocktail in convenient aliquots at -20°C.

• Benzonase^® Nuclease: Store at 4°C.

Wash buffer, Extraction Buffer I, Extraction Buffer II, Extraction Buffer III, Extraction Buffer IV, Protease Inhibitor Cocktail (Cat. No. 539134), Benzonase^® Nuclease (Cat. No. 71206), and a user protocol.

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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

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