The Thermo Scientific Subcellular Protein Fractionation Kit for Tissue includes all reagents and tissue strainers required to perform cell fractionation and enrichment of proteins from five different cellular compartments within 2 hours.
The complete Subcellular Protein Fractionation Kit for Tissue includes a unique 250µm tissue strainer to facilitate rapid separation of cell suspension from tissue debris after homogenization. The optimized reagents enable subsequent stepwise extraction of cytoplasmic, membrane, nuclear soluble, chromatin-bound and cytoskeletal proteins from the recovered cells with less than 15% cross-contamination. Tissue extracts obtained with this subcellular fractionation kit are compatible with a variety of downstream applications, including Western blotting, protein quantitation, electrophoretic mobility shift, reporter-gene and enzyme-activity assays.
- Optimized – formulations and protocols specific for fractionation of tissue
- Complete – obtain cytoplasmic, membrane, soluble nuclear, chromatin-bound and cytoskeletal protein fractions from a single kit
- Time efficient – extract functional protein fractions without ultracentrifugation in approx. 2 hours
- Convenient – unique tissue strainer allows rapid removal of tissue debris
- Compatible – use extracts for downstream applications such as protein assays, Western blotting, gel-shift assays and enzyme-activity assays
- Versatile – extract proteins from a variety of tissue types, including liver, heart, brain, kidney, lung and spleen
- Obtain cytoplasmic, soluble nuclear, chromatin-bound, membrane and cytoskeletal proteins from the same tissue sample
- Profile disease-state versus normal tissues for proteins from the cytoplasm, nucleus, membrane and cytoskeleton
- Study protein translocation in tissues
The Subcellular Protein Fractionation Kit for Tissue contains four extraction buffers, tissue strainers, a stabilized nuclease and Thermo Scientific Halt Protease Inhibitor Cocktail. Each kit has sufficient reagents and tissue strainers to process and fractionate 25 tissue samples up to 200mg each. The tissue is homogenized in the first reagent, which causes selective membrane permeabilization, releasing soluble cytoplasmic contents. This homogenate is transferred to a tissue strainer to remove excess tissue debris. 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.
|Schematic of the subcellular fractionation protocol. Proteins are sequentially extracted using differential detergents. CEB = cytoplasmic extraction buffer; MEB = membrane extraction buffer; NEB = nuclear extraction buffer; NEB + MNase = nuclear extraction buffer plus micrococcal nuclease (chromatin-bound fraction); PEB = pellet extraction buffer.
|Mouse tissue samples effectively fractionated using the Thermo Scientific Subcellular Protein Fractionation Kit for Tissues. Normalized loads of each extract (10µg) were analyzed by Western blotting. CE = cytoplasmic extract; ME = membrane extract; NE = nuclear extract; CB = chromatin-bound extract; PE = pellet extract.
Review of cell lysis and protein extraction
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