Proteins within a cell are localized to specific cellular compartments that provide crucial information about the function of the protein. The study of protein localization and translocation is increasingly important for understanding protein functions and diseases, especially cancers. Research is moving from cultured cell models to animal models for a more true representation of the biology. Tissue samples present an extra level of complexity with the tightly woven network of proteins and extracellular matrices. The Thermo Scientific Subcellular Protein Fractionation Kit for Tissues specifically addresses the fractionation of these elaborate structures. Subcellular protein fractionation of tissue samples enables protein localization discoveries, enhances detectability of low-abundance species, reduces sample complexity for proteomics and enables the monitoring of physiologic fluxes and redistribution under basal and stimulated/diseased conditions.

Protein translocation and localization to specific cellular compartments are important for normal cellular and disease events. Protein translocation is often assessed by isolating specific cellular compartments though differential ultracentrifugation or by sequential extractions of cellular components. As research trends from cell culture systems to animal models, the extraction of cellular components from tissue samples while minimizing contamination from other compartments has been challenging. Also, compared with cells, tissue homogenization has a higher level of complexity with the tightly woven network of proteins and extracellular matrices.

The Subcellular Protein Fractionation Kit for Tissues includes buffers formulated to efficiently extract protein from 50-200mg of tissue with minimal contamination between cellular compartments. Firstly, the tissue sample is homogenized in the cytoplasmic extraction buffer, which causes selective membrane permeabilization, releasing soluble cytoplasmic contents. This homogenate is then strained to remove excess debris. The second buffer 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 releases chromatin-bound nuclear proteins. The recovered insoluble pellet is then extracted with the final buffer to isolate cytoskeletal proteins (Figure 1).

Figure 1. Schematic of the subcellular fractionation procedure. 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.

The buffer extraction volumes have been optimized for effective protein concentrations in each fraction. The kit also includes a strainer for removing tissue debris after homogenization. The kit was validated using various mouse tissue samples. Protein localization was assessed for each tissue using ubiquitous, abundant or specific protein markers for each tissue type. Furthermore, proteins were assayed for functional activity in downstream assays.

Results and discussion

To evaluate fractionation efficiency, we performed the extraction procedure on various mouse tissue types, including heart, brain, kidney, lung, liver and spleen. To assess protein localization, we probed for compartment-specific protein markers by Western blotting. The results revealed minimal protein cross-contamination among the fractions (Figure 2), demonstrating that this method is useful for determining protein localization and translocation activities within tissues.

Figure 2. Fractions from multiple tissue samples resulted in minimal cross-contamination of target proteins. Mouse tissue samples were fractionated using the Subcellular Protein Fractionation Kit for Tissues. Normalized loads of each extract (10µg) were analyzed by Western blotting. (Data not shown for liver and spleen.) CE: cytoplasmic extract; ME: membrane extract; NE: nuclear extract; CB: chromatin-bound extract; PE: pellet extract.

To demonstrate protein functionality after subcellular fractionation, we performed an electrophoretic mobility shift assay (EMSA) using the nuclear extracts from mouse brain tissue and the Thermo Scientific LightShift Chemiluminescent DNA EMSA Kit. We used biotin-labeled DNA sequences known to bind to the transcription factor activator protein-1 (AP-1). This heterodimeric transcription factor regulates gene expression in response to a variety of stimuli including infections, stress, growth factors and cytokines. The AP-1 proteins from the nuclear extract bound to the specific DNA sequence, resulting in a molecular weight shift. These results show that the nuclear extract contained native, functional AP-1 proteins (Figure 3).

Figure 3. Nuclear extract from brain tissue is functional in an electrophoretic mobility-shift assay (EMSA). Mouse brain (180mg) was fractionated using the Subcellular Protein Fractionation Kit for Tissues. The resulting nuclear extract was used to perform an EMSA. Biotin-labeled and competitor (non-labeled) DNA duplexes were used to perform the reactions.

The Subcellular Protein Fractionation Kit for Tissues was specifically developed for the unique structure of many different tissue types, such as heart, kidney, brain, liver, spleen and lung. Using this kit, we were able to effectively extract five cellular compartments (i.e., cytoplasm, membrane, nuclear, chromatin-bound and cytoskeleton) from a single tissue sample without using ultra-centrifugation. Once extracted, these proteins were functional in downstream assays, such as Western blotting and DNA EMSA.


Subcellular protein fractionation: Mouse tissue was excised, weighed and rinsed in phosphate-buffered saline (PBS). The remaining steps were performed according to the instructions for the Subcellular Protein Fractionation Kit for Tissues (Part No. 87790). The tissue was first homogenized in the Cytoplasmic Extraction Buffer (CEB) using a tissue-tearor (BioSpec Products) or a Dounce tissue grinder. The homogenate was transferred to a Thermo Scientific Pierce Tissue Strainer in a 15mL conical tube and centrifuged for 5 minutes at 500 x g. The strainer with debris was discarded, and the supernatant (cytoplasmic extract) was recovered. The remaining pellet was resuspended in Membrane Extraction Buffer (MEB) and incubated at 4°C for 10 minutes with gentle mixing. The membrane extract was recovered by centrifugation at 3000 x g for 5 minutes. The pellet was resuspended in Nuclear Extraction Buffer (NEB) and incubated at 4°C for 30 minutes with gentle mixing. The soluble nuclear extract was separated by centrifugation at 5000 x g for 5 minutes. NEB containing micrococcal nuclease was added to the pellet and incubated at 37°C for 15 minutes with gentle mixing. Chromatin-bound nuclear proteins were released and recovered by centrifugation of 16,000 x g for 5 minutes. The remaining pellet was resuspended in Pellet Extraction Buffer (PEB) and incubated at room temperature for 10 minutes. The pellet extract was recovered by centrifugation of 16,000 x g for 5 minutes.

Western blot analysis: Protein concentration of each extract was determined, equal amounts of total protein (10µg) were resolved on denaturing 4-20% Tris-glycine SDS-polyacrylamide gels, and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with Thermo Scientific StartingBlock T20 (TBS) Blocking Buffer (Part No.37543) and incubated with primary antibody overnight at 4°C. Membranes were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Part No. 31430)(Part No. 31460). Bands were detected using Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080).

Primary antibodies: The following Thermo Scientific Primary Antibodies were used: anti-heat-shock protein 90 (HSP90) (Ab No. MA5-14866); anti-sodium-potassium ATPase (Ab No. PA5-17251); and anti-vascular endothelial growth factor receptor 2 (VEGF-R2) (Ab No. MA5-15157). Anti-histone deacetylase 2 (HDAC2) and anti-vimentin were obtained from Cell Signaling Technology. Anti-histone H3 was obtained from Santa Cruz Biotechnology. Anti-nucleoporin p62 was obtained from BD Biosciences.

Electrophoretic mobilized shift assay (EMSA): Mouse brain was excised, weighed, and rinsed in PBS. The brain was tprocessed for subcellular protein fractionation as previously stated. The soluble nuclear fraction was pre-cleared with Thermo Scientific High Capacity Streptavidin Agarose (Part No. 20357) to remove endogenous biotin. The reactions were performed using the LightShift Chemiluminescent EMSA Kit (Part No. 20148). Mouse brain nuclear extract (5µg) was incubated with either biotin-labeled or unlabeled (competitor; 100-fold molar excess) AP-1 target DNA duplexes and supplemented with 2.5% glycerol, 5mM magnesium chloride and 0.05% NP-40. Reactions were electrophoresed on a 6% polyacrylamide gel and transferred to Thermo Scientific Biodyne B Nylon Membrane (Part No. 77016). Detection was performed using the Thermo Scientific Chemiluminescent Nucleic Acid Detection Module (Part No. 89880).

Animal care:  C57BL/J6 mice (8-12 weeks old, mixed gender) from Charles River Laboratories and housed in the animal facility at the University of Illinois College of Medicine at Rockford, were used to obtain mouse tissue. Experiments were performed exactly as approved by the Animal Care and Use Committee at the University of Illinois, College of Medicine at Rockford and conducted in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals.