Subcellular Protein Fractionation Kit for
The Thermo Scientific Subcellular Protein Fractionation Kit for Cultured Cells enables segregation and enrichment of proteins from five different cellular compartments.
The kit enables the stepwise separation and extraction of cytoplasmic, membrane, nuclear soluble, chromatin-bound and cytoskeletal proteins from mammalian cultured cells. Extracts obtained with the Subcellular Protein Fractionation Kit for Cultured Cells are compatible with a variety of downstream applications including Western blotting, protein assays, electrophoretic mobility shift assays and reporter-gene and enzyme-activity assays.
Fractionation of subcellular proteins enables protein localization assessment and protein enrichment from specific cellular compartments. The 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.
Concise – extract functional cytoplasmic, membrane, nuclear soluble, chromatin-bound and cytoskeletal protein fractions 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, gel-shift assays 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
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 20µL 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.