Fractionate subcellular proteins for localization studies
Segregate and enrich proteins from five cellular compartments

The Thermo Scientific Subcellular Protein Fractionation 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 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.

Overview of the Subcellular Protein Fractionation Kit protocol

The Subcellular Protein Fractionation Kit 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 2 g of cell paste. The first reagent added to a cell pellet causes selective membrane permeablization, 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.

Schematic overview of the subcellular fractionation procedure. Cellular compartments are sequentially extracted by incubating cells with cytoplasmic extraction buffer (CEB) followed by membrane extraction buffer (MEB) and nuclear extraction buffer (NEB). Adding micrococcal nuclease (MNase) to NEB extracts chromatin-bound proteins from the cell pellet before adding the pellet extraction buffer (PEB) to solubilize cytoskeletal proteins.

Highlights of the Thermo Scientific Subcellular Fractionation Kit:

• 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
• Compatible – use extracts for downstream applications such as protein assays, Western blotting, gel-shift assays and enzyme activity assays

Applications for subcelluar fractionation:

• Extract and enrich proteins from different cellular compartments
• Separate and profile protein from the cytoplasm, nucleus, membrane and cytoskeleton from the same cell sample
• Study protein translocation

Cytoplasmic			HSP90	Western blots of fractionated cellular proteins. HeLa cells (2 x 10^6) were fractionated using the Subcellular Protein Fractionation Kit. Normalized portions of each extract (10 µg) were analyzed by Western blotting using specific antibodies against proteins from various cellular compartments including cytoplasmic (HSP90), plasma membrane (EGFR), endoplasmic reticulum (calreticulin), nuclear soluble (SP1 and HDAC2); chromatin-bound (histone 3); and cytoskeleton (cytokeratin 18 and vimentin). The blots were probed with goat anti-rabbit (H+L) HRP or goat anti-mouse (H+L) HRP and detected with Thermo Scientific SuperSignal West Dura Chemiluminescent Substrate (Product #34076). The results demonstrate clear fractionation of each protein with low cross contamination among fractions. CE: cytoplasmic extract, ME: membrane extract, NE: nuclear extract, CB: chromatin-bound extract, PE: pellet extract.
Membrane			EGFR
			calrecticulin
Nuclear  Soluble			SP1
			HDAC2
Chromatin  Bound			histone 3
Cytoskeletal			vimentin
			cytokeratin 18

Western blot analysis of protein translocation. Panel A. A549 cells (2 x 10^6) were either mock-treated (-) or incubated (+) with 20 µg/ml TNFα for 20 minutes and fractionated using the Subcellular Protein Fractionation Kit. Each extract (10 µg) was analyzed by Western blot using an anti-NFκB p65 antibody. Panel B. Serum-starved HeLa cells (2 x 10^6) were either mock-treated (-) or incubated (+) with 1 µM PMA for 20 minutes and fractionated. Each extract (10 µg) was analyzed by Western blot using an anti-PKCα antibody. Goat anti-rabbit or anti-mouse (H+L) HRP was used as the secondary antibody, and SuperSignal West Dura Chemiluminescent Substrate (Product #34076) was used for detection.

Related Products and Links:
MEM-PER Membrane Protein Extraction Reagent Kit
NE-PER Nuclear and Cytoplasmic Extraxtion Reagent Kit
Pierce Organelle Isolation Kits
Halt Protease Inhibitors and Phosphatase Inhibitors