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Cell Fractionation and Organelle Isolation
Cell fractionation kits are optimized for stepwise separation, enrichment, and extraction of proteins from different cellular fractions, including cytoplasmic, membrane, nuclear, chromatin-bound, and cytoskeletal proteins in 1–3 hours. Our organelle kits are optimized for the isolation and enrichment of organelles, including mitochondria, lysosomes, and synaptosomes. These reagents and kits provide high protein yield and enable consistent results due to our controls over ingredient quality and formulation.
Cell fractionation kits and reagents
| Nucleus, cytoplasm | Nucleus, cytoplasm, membrane, cytoskeletal, chromatin-bound | Synaptic proteins | |
|---|---|---|---|
| NE-PER Nuclear and Cytoplasmic Extraction Reagents | Subcellular Protein Fractionation Kits (cells or tissues) | Syn-PER Synaptic Protein Extraction Reagent | |
| Compatible sample types | Tissues and cultured mammalian cells | Tissues or cultured mammalian cells | Brain tissue and primary neurons |
| Sample processing time | 2 hours | 2–3 hours | <1 hour |
| Mechanical disruption required? | Yes, for tissue | Yes, for tissue | Yes, for tissue |
| Amount of sample processed | 50 samples with 2 million cells (20 µL packed) | 25 extractions of 200 mg tissue or 50 samples with 2 million cells (20 µL packed) | 10 g tissue or 500 x 35 mm dishes of primary cultured neurons |
| Protein assay compatibility | BCA Protein Assay (CER dilute 1:4), Bradford Assays (CER dilute 1:4) | BCA Protein Assay (all fractions), Bradford Assays (NEB fraction) | BCA Protein Assay |
| Downstream compatibility | Western blot, ELISA, EMSA, reporter assays, enzyme assays, amine reactive labeling CER fraction only: RNA EMSA, kinase assays, RT-PCR | IP, western blot, ELISA, EMSA, reporter assays, enzyme assays, amine reactive labeling | Neurotransmitter release assays, enzyme assays, immunoassays, chromatography, electrophoresis |
| Protease or phosphatase inhibitors recommended? | Yes | Protease inhibitors included in kit | Yes |
| Cat. No. | 78833 (15 mL) 78835 (75 mL) | 78840 (Cultured cells kit) 87790 (Tissue kit) | 87793 (100 mL) |
Nuclear protein extraction
A variety of methods exist to isolate nuclei and prepare nuclear protein extracts. However, most of these are lengthy processes requiring mechanical homogenization, freeze/thaw cycles, extensive centrifugation, or dialysis steps that may compromise the integrity of many fragile nuclear proteins. The NE-PER Nuclear and Cytoplasmic Extraction Kit enables a stepwise lysis of cells that generates both functional cytoplasmic and nuclear protein fractions in less than two hours.
Click image to enlarge
Figure 1. Total protein profile of cytoplasmic and nuclear extracts prepared from different mammalian cell lines. Mammalian cells were harvested, rinsed with PBS, and lysed using the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit. Extracts were quantified using the Pierce 660nm Protein Assay Reagent (Cat. No. 22660). Values are the average of two separate isolations.
Click image to enlarge
Figure 2. Minimize cross-contamination between cell fractions with NE-PER Nuclear and Cytoplasmic Extraction reagents. Nuclear and cytosolic fractions are obtained with minimal cross-contamination. HeLa cells were extracted with the Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Reagents or with nuclear extraction kits from other vendors. Samples of the nuclear and cytosolic fractions were analyzed by western blot using antibodies against common nuclear, cytoplasmic, and membrane protein markers. Nuclear fractions produced with the NE-PER kit had minimal to no contamination with cytosolic or membrane proteins.
Subcellular fractionation
Separation of proteins by subcellular localization is one of the methods to enrich for proteins while maintaining some biological context. Differential detergent extraction is a classic biochemical technique used to separate proteins based on sequential solubilization of cellular compartments using different detergents. The Thermo Scientific Subcellular Protein Fractionation Kit provides a combination of reagents for stepwise lysis of cells into cytoplasmic, membrane, nuclear-soluble, chromatin-bound, and cytoskeletal protein fractions that are functional.
Click image to enlarge
Figure 3. 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.
Organelle isolation kits
| Mitochondria | Mitochondria | Lysosome | Synaptosomes | |
|---|---|---|---|---|
 |  |  |  | |
| Mitochondria isolation kit for tissue | Mitochondria isolation kit for cultured cells | Lysosome enrichment kit for tissues and cultured cells | Syn-PER synaptic protein extraction reagent | |
| Compatible sample types | Heart and liver tissue | Cultured mammalian cells | Tissue and cultured cells | Tissue or primary cultured neurons |
| Sample processing time | 1 hour | 40 mins | 2 hours | <1 hour |
| Mechanical disruption required? | Dounce optional | Dounce optional | Yes: Dounce, sonication, or polytron tissue tearer | Yes: Dounce |
| Amount of sample processed | 50 samples each containing 50–200 mg of soft or hard tissue | 50 samples each containing 20 million cultured mammalian cells | 25 samples each containing 50–200 mg of cells or tissue | 10 g of neuronal tissue and primary cultured neurons per 100 mL |
| Protein assay compatibility | BCA Protein Assays, Bradford Assays | BCA Protein Assays, Detergent Compatible Bradford | Bradford Assays | BCA Protein Assays |
| Downstream compatibility | Western blot, ELISA, amine reactive labeling, apoptosis, signal transduction, and metabolic studies | Western blot, ELISA, amine reactive labeling, apoptosis, signal transduction, and metabolic studies | Western blot, 2D/MS, electron microscopy, disease profiling, gene expression, signal transduction, and interaction or localization studies | Western blot, enzymatic activity assays, protein-protein interaction studies, immunoprecipitations, neurotransmitter release study |
| Protease or phosphatase inhibitors recommended? | Yes, both | Yes, both | Yes, EDTA free | Yes, EDTA free |
| Cat. No | 89801 | 89874 | 89839 | 87793 |
Retain activity while enriching key subcellular components
Figure 4. Integrity of mitochondria extracted from cultured cells. C6 cells were extracted with Thermo Scientific Mitochondria Isolation Kit using the reagent-based method (A and B) or Dounce homogenization (C and D). Mitochondrial (M) and cytosolic (C) fractions were analyzed via western blot for cytochrome C (A and C) or voltage-dependent anion channel (VDAC) (B and D). Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate (Cat. No. 34580) was used for detection. Results indicate that mitochondria remained intact.
Figure 5. Greater enrichment of synaptic proteins is achieved in samples prepared using the Thermo Scientific Syn-PER Reagent than with a homebrew reagent. Total protein (10 µg) from mouse brain tissue homogenates (H), cytosol (C) fraction, and synaptosome (Syn) suspension were analyzed by western blot. The total protein yield in the synaptosome suspension produced from fresh mouse brain (200 mg) was three-fold greater using the Syn-PER Reagent compared to a typical homebrew reagent.
Click image to enlarge
Product manuals
Application notes
Cell fractionation kits and reagents
| Nucleus, cytoplasm | Nucleus, cytoplasm, membrane, cytoskeletal, chromatin-bound | Synaptic proteins | |
|---|---|---|---|
| NE-PER Nuclear and Cytoplasmic Extraction Reagents | Subcellular Protein Fractionation Kits (cells or tissues) | Syn-PER Synaptic Protein Extraction Reagent | |
| Compatible sample types | Tissues and cultured mammalian cells | Tissues or cultured mammalian cells | Brain tissue and primary neurons |
| Sample processing time | 2 hours | 2–3 hours | <1 hour |
| Mechanical disruption required? | Yes, for tissue | Yes, for tissue | Yes, for tissue |
| Amount of sample processed | 50 samples with 2 million cells (20 µL packed) | 25 extractions of 200 mg tissue or 50 samples with 2 million cells (20 µL packed) | 10 g tissue or 500 x 35 mm dishes of primary cultured neurons |
| Protein assay compatibility | BCA Protein Assay (CER dilute 1:4), Bradford Assays (CER dilute 1:4) | BCA Protein Assay (all fractions), Bradford Assays (NEB fraction) | BCA Protein Assay |
| Downstream compatibility | Western blot, ELISA, EMSA, reporter assays, enzyme assays, amine reactive labeling CER fraction only: RNA EMSA, kinase assays, RT-PCR | IP, western blot, ELISA, EMSA, reporter assays, enzyme assays, amine reactive labeling | Neurotransmitter release assays, enzyme assays, immunoassays, chromatography, electrophoresis |
| Protease or phosphatase inhibitors recommended? | Yes | Protease inhibitors included in kit | Yes |
| Cat. No. | 78833 (15 mL) 78835 (75 mL) | 78840 (Cultured cells kit) 87790 (Tissue kit) | 87793 (100 mL) |
Nuclear protein extraction
A variety of methods exist to isolate nuclei and prepare nuclear protein extracts. However, most of these are lengthy processes requiring mechanical homogenization, freeze/thaw cycles, extensive centrifugation, or dialysis steps that may compromise the integrity of many fragile nuclear proteins. The NE-PER Nuclear and Cytoplasmic Extraction Kit enables a stepwise lysis of cells that generates both functional cytoplasmic and nuclear protein fractions in less than two hours.
Click image to enlarge
Figure 1. Total protein profile of cytoplasmic and nuclear extracts prepared from different mammalian cell lines. Mammalian cells were harvested, rinsed with PBS, and lysed using the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit. Extracts were quantified using the Pierce 660nm Protein Assay Reagent (Cat. No. 22660). Values are the average of two separate isolations.
Click image to enlarge
Figure 2. Minimize cross-contamination between cell fractions with NE-PER Nuclear and Cytoplasmic Extraction reagents. Nuclear and cytosolic fractions are obtained with minimal cross-contamination. HeLa cells were extracted with the Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Reagents or with nuclear extraction kits from other vendors. Samples of the nuclear and cytosolic fractions were analyzed by western blot using antibodies against common nuclear, cytoplasmic, and membrane protein markers. Nuclear fractions produced with the NE-PER kit had minimal to no contamination with cytosolic or membrane proteins.
Subcellular fractionation
Separation of proteins by subcellular localization is one of the methods to enrich for proteins while maintaining some biological context. Differential detergent extraction is a classic biochemical technique used to separate proteins based on sequential solubilization of cellular compartments using different detergents. The Thermo Scientific Subcellular Protein Fractionation Kit provides a combination of reagents for stepwise lysis of cells into cytoplasmic, membrane, nuclear-soluble, chromatin-bound, and cytoskeletal protein fractions that are functional.
Click image to enlarge
Figure 3. 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.
Organelle isolation kits
| Mitochondria | Mitochondria | Lysosome | Synaptosomes | |
|---|---|---|---|---|
| | | | |
| Mitochondria isolation kit for tissue | Mitochondria isolation kit for cultured cells | Lysosome enrichment kit for tissues and cultured cells | Syn-PER synaptic protein extraction reagent | |
| Compatible sample types | Heart and liver tissue | Cultured mammalian cells | Tissue and cultured cells | Tissue or primary cultured neurons |
| Sample processing time | 1 hour | 40 mins | 2 hours | <1 hour |
| Mechanical disruption required? | Dounce optional | Dounce optional | Yes: Dounce, sonication, or polytron tissue tearer | Yes: Dounce |
| Amount of sample processed | 50 samples each containing 50–200 mg of soft or hard tissue | 50 samples each containing 20 million cultured mammalian cells | 25 samples each containing 50–200 mg of cells or tissue | 10 g of neuronal tissue and primary cultured neurons per 100 mL |
| Protein assay compatibility | BCA Protein Assays, Bradford Assays | BCA Protein Assays, Detergent Compatible Bradford | Bradford Assays | BCA Protein Assays |
| Downstream compatibility | Western blot, ELISA, amine reactive labeling, apoptosis, signal transduction, and metabolic studies | Western blot, ELISA, amine reactive labeling, apoptosis, signal transduction, and metabolic studies | Western blot, 2D/MS, electron microscopy, disease profiling, gene expression, signal transduction, and interaction or localization studies | Western blot, enzymatic activity assays, protein-protein interaction studies, immunoprecipitations, neurotransmitter release study |
| Protease or phosphatase inhibitors recommended? | Yes, both | Yes, both | Yes, EDTA free | Yes, EDTA free |
| Cat. No | 89801 | 89874 | 89839 | 87793 |
Retain activity while enriching key subcellular components
Figure 4. Integrity of mitochondria extracted from cultured cells. C6 cells were extracted with Thermo Scientific Mitochondria Isolation Kit using the reagent-based method (A and B) or Dounce homogenization (C and D). Mitochondrial (M) and cytosolic (C) fractions were analyzed via western blot for cytochrome C (A and C) or voltage-dependent anion channel (VDAC) (B and D). Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate (Cat. No. 34580) was used for detection. Results indicate that mitochondria remained intact.
Figure 5. Greater enrichment of synaptic proteins is achieved in samples prepared using the Thermo Scientific Syn-PER Reagent than with a homebrew reagent. Total protein (10 µg) from mouse brain tissue homogenates (H), cytosol (C) fraction, and synaptosome (Syn) suspension were analyzed by western blot. The total protein yield in the synaptosome suspension produced from fresh mouse brain (200 mg) was three-fold greater using the Syn-PER Reagent compared to a typical homebrew reagent.
Click image to enlarge
Product manuals
Application notes
Related products and downstream applications
For Research Use Only. Not for use in diagnostic procedures.