Pierce Cell Fractionation kit- NE-PER Nuclear and Cytoplasmic extraction kit

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.

Membrane Protein Isolation   Total Cell Lysis Buffers

Cell fractionation kits and reagents

 Nucleus, cytoplasmNucleus, cytoplasm, membrane, cytoskeletal, chromatin-boundSynaptic proteins
 NE-PER Nuclear and Cytoplasmic Extraction ReagentsSubcellular Protein Fractionation Kits (cells or tissues)Syn-PER Synaptic Protein Extraction Reagent
Compatible sample typesTissues and cultured mammalian cellsTissues or cultured mammalian cellsBrain tissue and primary neurons
Sample processing time2 hours2–3 hours<1 hour
Mechanical disruption required?Yes, for tissueYes, for tissueYes, 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)10 g tissue or 500 x 35 mm dishes of primary cultured neurons
Protein assay compatibilityBCA 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 compatibilityWestern 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 labelingNeurotransmitter release assays, enzyme assays, immunoassays, chromatography, electrophoresis
Protease or phosphatase inhibitors recommended?YesProtease inhibitors included in kitYes
Cat. No.78833 (15 mL)
78835 (75 mL)
78840 (Cultured cells kit)
87790 (Tissue kit)
87793 (100 mL)

Discover kits for membrane protein extraction and isolation

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.

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.

Organelle isolation kits

 MitochondriaMitochondriaLysosomeSynaptosomes
 
 Mitochondria isolation kit for tissueMitochondria isolation kit for cultured cellsLysosome enrichment kit for tissues and cultured cellsSyn-PER synaptic protein extraction reagent
Compatible sample typesHeart and liver tissueCultured mammalian cellsTissue and cultured cellsTissue or primary cultured neurons
Sample processing time1 hour40 mins2 hours<1 hour
Mechanical disruption required?Dounce optionalDounce optionalYes: Dounce, sonication, or polytron tissue tearerYes: Dounce
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 tissue10 g of neuronal tissue and primary cultured neurons per 100 mL
Protein assay compatibilityBCA Protein Assays, Bradford AssaysBCA Protein Assays, Detergent Compatible BradfordBradford AssaysBCA Protein 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 studiesWestern blot, enzymatic activity assays, protein-protein interaction studies, immunoprecipitations, neurotransmitter release study
Protease or phosphatase inhibitors recommended?Yes, bothYes, bothYes, EDTA freeYes, EDTA free
Cat. No89801898748983987793

Retain activity while enriching key subcellular components

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 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.

Cell fractionation kits and reagents

 Nucleus, cytoplasmNucleus, cytoplasm, membrane, cytoskeletal, chromatin-boundSynaptic proteins
 NE-PER Nuclear and Cytoplasmic Extraction ReagentsSubcellular Protein Fractionation Kits (cells or tissues)Syn-PER Synaptic Protein Extraction Reagent
Compatible sample typesTissues and cultured mammalian cellsTissues or cultured mammalian cellsBrain tissue and primary neurons
Sample processing time2 hours2–3 hours<1 hour
Mechanical disruption required?Yes, for tissueYes, for tissueYes, 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)10 g tissue or 500 x 35 mm dishes of primary cultured neurons
Protein assay compatibilityBCA 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 compatibilityWestern 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 labelingNeurotransmitter release assays, enzyme assays, immunoassays, chromatography, electrophoresis
Protease or phosphatase inhibitors recommended?YesProtease inhibitors included in kitYes
Cat. No.78833 (15 mL)
78835 (75 mL)
78840 (Cultured cells kit)
87790 (Tissue kit)
87793 (100 mL)

Discover kits for membrane protein extraction and isolation

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.

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.

Organelle isolation kits

 MitochondriaMitochondriaLysosomeSynaptosomes
 
 Mitochondria isolation kit for tissueMitochondria isolation kit for cultured cellsLysosome enrichment kit for tissues and cultured cellsSyn-PER synaptic protein extraction reagent
Compatible sample typesHeart and liver tissueCultured mammalian cellsTissue and cultured cellsTissue or primary cultured neurons
Sample processing time1 hour40 mins2 hours<1 hour
Mechanical disruption required?Dounce optionalDounce optionalYes: Dounce, sonication, or polytron tissue tearerYes: Dounce
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 tissue10 g of neuronal tissue and primary cultured neurons per 100 mL
Protein assay compatibilityBCA Protein Assays, Bradford AssaysBCA Protein Assays, Detergent Compatible BradfordBradford AssaysBCA Protein 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 studiesWestern blot, enzymatic activity assays, protein-protein interaction studies, immunoprecipitations, neurotransmitter release study
Protease or phosphatase inhibitors recommended?Yes, bothYes, bothYes, EDTA freeYes, EDTA free
Cat. No89801898748983987793

Retain activity while enriching key subcellular components

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 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.

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