Subcellular Protein Fractionation Kit for Cultured Cells

Thermo Scientific Subcellular Protein Fractionation Kit for Cultured Cells enables segregation and enrichment of proteins from five different cellular compartments.

Features of Subcellular Protein Fractionation Kit for Cultured Cells:

• Concise—extract functional cytoplasmic, membrane, nuclear soluble, chromatin-bound and cytoskeletal protein fractions from mammalian cultured cells in less than 3 hours
• Convenient—perform a simple procedure without using ultracentrifugation over gradients
• Complete—obtain cytoplasmic, membrane, soluble nuclear, chromatin-bound and cytoskeletal protein fractions from a single kit
• Cultured Cells—developed and optimized for use with cultured mammalian cells; for tissue samples use the Subcellular Fractionation Kit for Tissues
• Compatible—use extracts for downstream applications such as protein assays, Western blotting, protein assays, electrophoretic mobility shift assays, reporter-gene and enzyme-activity assays.

Applications for subcellular fractionation:
• Extract and enrich proteins from different cellular compartments
• Isolate cytoplasmic, nuclear, membrane and cytoskeletal proteins from the same cells
• Profile cells for proteins from the cytoplasm, nucleus, membrane and cytoskeleton
• Study protein translocation

Fractionation of subcellular proteins enables protein localization assessment and protein enrichment from specific cellular compartments. Thermo Scientific Subcellular Protein Fractionation Kit includes a combination of reagents for stepwise lysis of cells into functional cytoplasmic, membrane, nuclear soluble, chromatin-bound and cytoskeletal protein fractions in less than 3 hours. Extracts from each subcellular compartment generally have less than 15% contamination between fractions, which is sufficient purity for most experiments studying protein localization and redistribution.

The Subcellular Protein Fractionation Kit for Cultured Cells contains four extraction buffers, a stabilized nuclease and Thermo Scientific Halt Protease Inhibitor Cocktail. Each kit has enough reagents to fractionate 50 cell pellets of 20 μL each, equivalent to approximately 2g of cell paste. (For tissue samples, use our Subcellular Protein Fractionation Kit for Tissue.)

The first kit reagent, when added to a cell pellet, causes selective membrane permeabilization, releasing soluble cytoplasmic contents. The second reagent dissolves plasma, mitochondria and ER-golgi membranes but does not solubilize the nuclear membranes. After recovering intact nuclei by centrifugation, a third reagent yields the soluble nuclear extract. An additional nuclear extraction with micrococcal nuclease is performed to release chromatin-bound nuclear proteins. The recovered insoluble pellet is then extracted with the final reagent to isolate cytoskeletal proteins.

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