Cell fractionation and organelle isolation are essential techniques in biological research involved the separation of cellular components for downstream assays including western blotting, protein assays, and enzyme activity assays. Cell fractionation is the breakdown of cells into distinct components such as cytoplasmic, membrane, nuclear, chromatin-bound, and cytoskeletal proteins. On the other hand, organelle isolation focuses on the separation of subcellular structures like mitochondria, lysozyme, and synaptosomes. These methods allow for the comprehensive analysis of cellular structures along with enabling a deeper understanding of cellular functions and interactions within the complex microenvironment of the cell.

Thermo Fisher Scientific offers cell fractionation and organelle isolation kits and reagents that are optimized for stepwise separation, enrichment, and extraction of proteins. These products are specifically designed for quick and efficient fractionation or isolation of cells and tissues, resulting in high protein yields within 1-3 hours.  Choose from our comprehensive range of cell fractionation or organelle isolation product based on your specific separation needs. 

Learn more: Cell Fractionation and Organelle Isolation

Choose the optimal fractionation and isolation reagent

 nuclear and cytoplasmic extraction kitprotein fraction kitprotein fractionation kit
 NE-PER Nuclear and Cytoplasmic Extraction ReagentsSubcellular Protein Fractionation Kit for cultured cellsSubcellular Protein Fractionation Kit for tissues
When to use (compatible cell structure)Nucleus, cytoplasmNucleus, cytoplasm, membrane, cytoskeletal, chromatin-bound
Compatible sample typesTissues and cultured mammalian cellsTissues or cultured mammalian cells
Sample processing time2 hours2–3 hours

Mechanical disruption required?

Yes, for tissue

Yes, for tissue

Amount of sample processed50 samples with 2 million cells (20 µL packed)25 extractions of 200 mg tissue or 50 samples with 2 million cells (20 µL packed)
Compatible protein assaysBCA Protein Assay (CER dilute 1:4), Bradford Assays (CER dilute 1:4)BCA Protein Assay (all fractions), Bradford Assays (NEB fraction)
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
Protease or phosphatase inhibitors recommended?YesProtease inhibitors included in kit
Available size(s)15 mL
75 mL
1 kit (50 samples)1 kit (25 samples)
User guideUser Guide: NE-PER Nuclear and Cytoplasmic Extraction ReagentsUser Guide: Subcellular Protein Fractionation Kit for Cultured CellUser Guide: Subcellular Protein Fractionation Kit for Tissue


Cell fractionation kits and reagents

Nuclear and cytoplasmic 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 generates both functional cytoplasmic and nuclear protein fractions recovered from the same cell (Figure 1) or tissue (Figure 2) sample in less than two hours. The simple protocol involves a stepwise lysis that separates the cytoplasm from intact nuclei, and then extracts nuclear protein away from genomic DNA and mRNA. Yield from two million cells typically includes 200 to 500 µg of cytoplasmic protein and 100 to 200 µg of nuclear protein (at a concentration of 1 mg/mL). Additionally, cross-contamination between cytoplasmic and nuclear fractions has been shown to be less than 10% (data not shown).

Subcellular protein fractionation kit for cultured cells or tissues

Separation of proteins by subcellular isolation is one of the methods to enrich for proteins while maintaining the ability to characterize biological proteins and study protein translocation. 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 (Figure3, 4). Cross-contamination between extracts from each subcellular fractions is generally less than 15% (data not shown).

 Learn more: Subcellular protein fractionation to enhance proteomic coverage of cultured cells

Figure 4. 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 (Part No. 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

 
 Mitochondria isolation kit for tissueMitochondria isolation kit for cultured cellsLysosome enrichment kit for tissues and cultured cells
When to use (compatible cell organelle)MitochondriaLysosome
Compatible sample typesHeart and liver tissueCultured mammalian cellsTissue and cultured cells
Sample processing time1 hour40 mins2 hours
Mechanical disruption required?Dounce optionalDounce optionalYes: Dounce, sonication, or polytron tissue tearer
Amount of sample processed50 samples each containing 50–200 mg of soft or hard tissue50 samples each containing 20 million cultured mammalian cells25 samples each containing 50–200 mg of cells or tissue
Compatible protein assaysBCA Protein Assays, Bradford AssaysBCA Protein Assays, Detergent Compatible BradfordBradford Assays
Downstream compatibilityWestern blot, ELISA, amine reactive labeling, apoptosis, signal transduction, and metabolic studiesWestern blot, ELISA, amine reactive labeling, apoptosis, signal transduction, and metabolic studiesWestern blot, 2D/MS, electron microscopy, disease profiling, gene expression, signal transduction, and interaction or localization studies
Protease or phosphatase inhibitors recommended?Yes, bothYes, bothYes, EDTA free
Available size(s)1 kit (50 isolations)1 kit (50 isolations)1 kit (25 samples of 50 to 200 mg cells or tissues each)
User guideUser Guide: Mitochondria Isolation Kit for TissueUser Guide: Mitochondria Isolation Kit for Cultured CellsUser Guide: Lysosome Enrichment Kit for Tissue and Cultured Cells


Organelle isolation kits

Mitochondria isolation kit for tissue and cultured cells

The mitochondria isolation kits for tissue or cultured cells include optimized reagents and protocols that enable high yields of pure mitochondria. Additionally, they each use differential centrifugation to separate intact mitochondria.

The Mitochondria Isolation Kit for Tissue offers two streamlined methods, a reagent-based or Dounce homogenization procedure, to isolate mitochondria from soft and hard tissue samples in about an hour (Figure 5). The reagent-based option enables the simultaneous processing of multiple samples.

The Mitochondria Isolation Kit for Cultured Cells primarily uses a reagent-based method to isolate mitochondria in approximately 40 minutes (Figure 6). Like the mitochondria isolation kit for tissues, this reagent-based method can be used to process multiple samples at the same time. If greater yield is needed, the mitochondria isolation kit for cultured cells also offers an optimized Dounce homogenization procedure that results in 2-fold greater mitochondria recovery.

Mitochondria integrity is retained after isolation using Thermo Scientific Mitochondria isolation kit noted by expression of cytochrome C and VDAC in the isolated fraction in western blot analysis

Figure 5. Integrity of mitochondria extracted from tissues. Mitochondria (M) and cytosolic (C) fractions were prepared from fresh rat liver (Panels 1, 2, 3, 5 and 6) and heart (Panel 4) tissue samples using the reagent-based and Dounce homogenization methods of the Thermo Scientific Mitochondria Isolation Kit for Tissue. Fractions were analyzed via Western blot for COX4, VDAC and Cytochrome C mitochondrial proteins. Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate was used for detection. COX4 is an inner-mitochondria membrane protein, VDAC is an outer-mitochondria membrane protein and cytochrome C is located in the intermembrane space. Results indicate that cytosolic proteins were thoroughly separated from the isolated mitochondria.

western blot analysis of mitochondria and cytosolic fractions from cultured cells

Figure 6. 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 was used for detection. Results indicate that mitochondria remained intact.

Lysosome Enrichment Kit for Tissues and Cultured Cells

The Lysosome Enrichment Kit for Tissues and Cultured Cells allows for the isolation and enrichment of intact lysosomes from cells (Figure 7) and soft and hard tissue samples. Similar to the mitochondria isolation kits, the lysosome enrichment kit uses reagent-based or Dounce homogenization method. For the reagent-based procedure, the kit contains OptiPrep Cell Separation Media to isolate and enrich lysosomes from the contaminating cell structures.

 synaptic protein extraction reagent 
 Syn-PER Synaptic Protein Extraction Reagent
When to use (compatible cell organelle)Synaptic proteinsSynaptosomes
Compatible sample typesBrain tissue and primary cultured neurons
Sample processing time<1 hour
Mechanical disruption required?Yes, dounce for tissue
Amount of sample processed10 g neuronal tissue or primary cultured neurons per 100 mL
Compatible protein assaysBCA Protein Assays
Downstream compatibilitywestern blot, enzymatic activity assays, protein-protein interaction studies, immunoprecipitationsneurotransmitter release assay only
Recommended supplemental protease/phosphatase inhibitorsYes, EDTA free
Available size(s)100 mL
User guideUser Guide: Syn-PER Synaptic Protein Extraction Reagent


Synaptic protein extraction reagent

Synaptosomes are isolated nerve terminals that are generated during the homogenization of nerve tissue. Syn-PER Synaptic Protein Extraction Reagent is specifically formulated to gently and rapidly extract synaptosomes from neuronal tissue (Figure 8) and primary cultured neurons with high yield. Synaptosomes are commonly used to study synaptic function such as neurotransmitter release, identification of changes in protein composition and function in synapses, and protein phosphorylation events associated with synapses. 

The Syn-PER reagent can be used to isolate functional pre- and post-synaptic proteins (i.e., intact membranes and protein complexes of synapses) and allows for the preservation of phosphoprotein integrity. Although, this reagent does not contain protease or phosphatase inhibitors, Halt Protease and/or Phosphatase Inhibitors can be added before use to prevent proteolysis or for additional protection from the high phosphatase activity present in brain tissue.

Methods article: Method to isolate functional synaptosomes

Figure 8. Synaptic protein enrichment in brain tissue. 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.

 nuclear and cytoplasmic extraction kitprotein fraction kitprotein fractionation kit
 NE-PER Nuclear and Cytoplasmic Extraction ReagentsSubcellular Protein Fractionation Kit for cultured cellsSubcellular Protein Fractionation Kit for tissues
When to use (compatible cell structure)Nucleus, cytoplasmNucleus, cytoplasm, membrane, cytoskeletal, chromatin-bound
Compatible sample typesTissues and cultured mammalian cellsTissues or cultured mammalian cells
Sample processing time2 hours2–3 hours

Mechanical disruption required?

Yes, for tissue

Yes, for tissue

Amount of sample processed50 samples with 2 million cells (20 µL packed)25 extractions of 200 mg tissue or 50 samples with 2 million cells (20 µL packed)
Compatible protein assaysBCA Protein Assay (CER dilute 1:4), Bradford Assays (CER dilute 1:4)BCA Protein Assay (all fractions), Bradford Assays (NEB fraction)
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
Protease or phosphatase inhibitors recommended?YesProtease inhibitors included in kit
Available size(s)15 mL
75 mL
1 kit (50 samples)1 kit (25 samples)
User guideUser Guide: NE-PER Nuclear and Cytoplasmic Extraction ReagentsUser Guide: Subcellular Protein Fractionation Kit for Cultured CellUser Guide: Subcellular Protein Fractionation Kit for Tissue


Cell fractionation kits and reagents

Nuclear and cytoplasmic 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 generates both functional cytoplasmic and nuclear protein fractions recovered from the same cell (Figure 1) or tissue (Figure 2) sample in less than two hours. The simple protocol involves a stepwise lysis that separates the cytoplasm from intact nuclei, and then extracts nuclear protein away from genomic DNA and mRNA. Yield from two million cells typically includes 200 to 500 µg of cytoplasmic protein and 100 to 200 µg of nuclear protein (at a concentration of 1 mg/mL). Additionally, cross-contamination between cytoplasmic and nuclear fractions has been shown to be less than 10% (data not shown).

Subcellular protein fractionation kit for cultured cells or tissues

Separation of proteins by subcellular isolation is one of the methods to enrich for proteins while maintaining the ability to characterize biological proteins and study protein translocation. 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 (Figure3, 4). Cross-contamination between extracts from each subcellular fractions is generally less than 15% (data not shown).

 Learn more: Subcellular protein fractionation to enhance proteomic coverage of cultured cells

Figure 4. 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 (Part No. 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

 
 Mitochondria isolation kit for tissueMitochondria isolation kit for cultured cellsLysosome enrichment kit for tissues and cultured cells
When to use (compatible cell organelle)MitochondriaLysosome
Compatible sample typesHeart and liver tissueCultured mammalian cellsTissue and cultured cells
Sample processing time1 hour40 mins2 hours
Mechanical disruption required?Dounce optionalDounce optionalYes: Dounce, sonication, or polytron tissue tearer
Amount of sample processed50 samples each containing 50–200 mg of soft or hard tissue50 samples each containing 20 million cultured mammalian cells25 samples each containing 50–200 mg of cells or tissue
Compatible protein assaysBCA Protein Assays, Bradford AssaysBCA Protein Assays, Detergent Compatible BradfordBradford Assays
Downstream compatibilityWestern blot, ELISA, amine reactive labeling, apoptosis, signal transduction, and metabolic studiesWestern blot, ELISA, amine reactive labeling, apoptosis, signal transduction, and metabolic studiesWestern blot, 2D/MS, electron microscopy, disease profiling, gene expression, signal transduction, and interaction or localization studies
Protease or phosphatase inhibitors recommended?Yes, bothYes, bothYes, EDTA free
Available size(s)1 kit (50 isolations)1 kit (50 isolations)1 kit (25 samples of 50 to 200 mg cells or tissues each)
User guideUser Guide: Mitochondria Isolation Kit for TissueUser Guide: Mitochondria Isolation Kit for Cultured CellsUser Guide: Lysosome Enrichment Kit for Tissue and Cultured Cells


Organelle isolation kits

Mitochondria isolation kit for tissue and cultured cells

The mitochondria isolation kits for tissue or cultured cells include optimized reagents and protocols that enable high yields of pure mitochondria. Additionally, they each use differential centrifugation to separate intact mitochondria.

The Mitochondria Isolation Kit for Tissue offers two streamlined methods, a reagent-based or Dounce homogenization procedure, to isolate mitochondria from soft and hard tissue samples in about an hour (Figure 5). The reagent-based option enables the simultaneous processing of multiple samples.

The Mitochondria Isolation Kit for Cultured Cells primarily uses a reagent-based method to isolate mitochondria in approximately 40 minutes (Figure 6). Like the mitochondria isolation kit for tissues, this reagent-based method can be used to process multiple samples at the same time. If greater yield is needed, the mitochondria isolation kit for cultured cells also offers an optimized Dounce homogenization procedure that results in 2-fold greater mitochondria recovery.

Mitochondria integrity is retained after isolation using Thermo Scientific Mitochondria isolation kit noted by expression of cytochrome C and VDAC in the isolated fraction in western blot analysis

Figure 5. Integrity of mitochondria extracted from tissues. Mitochondria (M) and cytosolic (C) fractions were prepared from fresh rat liver (Panels 1, 2, 3, 5 and 6) and heart (Panel 4) tissue samples using the reagent-based and Dounce homogenization methods of the Thermo Scientific Mitochondria Isolation Kit for Tissue. Fractions were analyzed via Western blot for COX4, VDAC and Cytochrome C mitochondrial proteins. Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate was used for detection. COX4 is an inner-mitochondria membrane protein, VDAC is an outer-mitochondria membrane protein and cytochrome C is located in the intermembrane space. Results indicate that cytosolic proteins were thoroughly separated from the isolated mitochondria.

western blot analysis of mitochondria and cytosolic fractions from cultured cells

Figure 6. 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 was used for detection. Results indicate that mitochondria remained intact.

Lysosome Enrichment Kit for Tissues and Cultured Cells

The Lysosome Enrichment Kit for Tissues and Cultured Cells allows for the isolation and enrichment of intact lysosomes from cells (Figure 7) and soft and hard tissue samples. Similar to the mitochondria isolation kits, the lysosome enrichment kit uses reagent-based or Dounce homogenization method. For the reagent-based procedure, the kit contains OptiPrep Cell Separation Media to isolate and enrich lysosomes from the contaminating cell structures.

 synaptic protein extraction reagent 
 Syn-PER Synaptic Protein Extraction Reagent
When to use (compatible cell organelle)Synaptic proteinsSynaptosomes
Compatible sample typesBrain tissue and primary cultured neurons
Sample processing time<1 hour
Mechanical disruption required?Yes, dounce for tissue
Amount of sample processed10 g neuronal tissue or primary cultured neurons per 100 mL
Compatible protein assaysBCA Protein Assays
Downstream compatibilitywestern blot, enzymatic activity assays, protein-protein interaction studies, immunoprecipitationsneurotransmitter release assay only
Recommended supplemental protease/phosphatase inhibitorsYes, EDTA free
Available size(s)100 mL
User guideUser Guide: Syn-PER Synaptic Protein Extraction Reagent


Synaptic protein extraction reagent

Synaptosomes are isolated nerve terminals that are generated during the homogenization of nerve tissue. Syn-PER Synaptic Protein Extraction Reagent is specifically formulated to gently and rapidly extract synaptosomes from neuronal tissue (Figure 8) and primary cultured neurons with high yield. Synaptosomes are commonly used to study synaptic function such as neurotransmitter release, identification of changes in protein composition and function in synapses, and protein phosphorylation events associated with synapses. 

The Syn-PER reagent can be used to isolate functional pre- and post-synaptic proteins (i.e., intact membranes and protein complexes of synapses) and allows for the preservation of phosphoprotein integrity. Although, this reagent does not contain protease or phosphatase inhibitors, Halt Protease and/or Phosphatase Inhibitors can be added before use to prevent proteolysis or for additional protection from the high phosphatase activity present in brain tissue.

Methods article: Method to isolate functional synaptosomes

Figure 8. Synaptic protein enrichment in brain tissue. 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.


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