Nuclear cytosol fractionation kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Nuclear/Cytosol Fractionation Kit is a laboratory tool used to separate cellular components, specifically the nucleus and cytosol, for analysis. The kit provides a standardized process to extract and isolate these subcellular fractions from cell samples.

Automatically generated - may contain errors

Lab products found in correlation

β actin, by Cell Signaling Technology (3 mentions) Enhanced chemiluminescence method, by Bio-Rad (2 mentions) Imagequant las 500 imaging system, by GE Healthcare (2 mentions) Pvdf membrane, by Bio-Rad (2 mentions) Ecl kit, by Thermo Fisher Scientific (2 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Sybr premix ex taq 2, by Takara Bio (1 mentions) Primescript rt reagent kit, by Takara Bio (1 mentions) C myc, by GeneTex (1 mentions) Survivin, by Cell Signaling Technology (1 mentions) Vascular endothelial growth factor (vegf), by Cell Signaling Technology (1 mentions) Bcl 2, by Cell Signaling Technology (1 mentions) β catenin, by Cell Signaling Technology (1 mentions) Erk1 2, by Cell Signaling Technology (1 mentions) Trpm8, by Abcam (1 mentions) Pierce bca assay kit, by Thermo Fisher Scientific (1 mentions) Gapdh, by Merck Group (1 mentions) Enhanced chemi luminescence (ecl), by Elpis Biotech (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Chemiluminescence system, by Thermo Fisher Scientific (1 mentions)

11 protocols using nuclear cytosol fractionation kit

Comprehensive Analysis of RNA Extraction and Quantification in Osteosarcoma

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total RNA extraction was performed with the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from certain clinical tumor samples or osteosarcoma cells according to the manufacturer’s instructions. The extraction of cytoplasmic RNA and nuclear RNA from OS cells were performed using the Nuclear/Cytosol Fractionation Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The RNA samples were stored at -80 ?. The PrimeScript RT reagent kit (TaKaRa Bio Inc., Kusatsu, Shiga, Japan) and SYBR Premix Ex Taq II (TaKaRa Bio Inc., Kusatsu, Shiga, Japan) were used to analyze the mRNA and circRNA levels. The β-actin expression level was considered the reference standard against which the mRNA and circRNA levels were compared. The expression of circRNA and mRNA levels were calculated by using the 2^-∆∆Ct method. Nuclear circRNA expression was normalized against U6 expression. Primer sequences are shown in Supplementary Table 1.

Shen S., Xu Y., Gong Z., Yao T., Qiao D., Huang Y., Zhang Z., Gao J., Ni H., Jin Z., Zhu Y., Wu H., Wang Q., Fang X., Huang K, & Ma J. (2023). Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism. Aging and Disease, 14(2), 529-547.

+ Open protocol

+ Expand

Nuclear-Cytosolic Fractionation and lncRNA Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

According to the method of a report (Lin H. et al., 2020 (link)) and the instructions of the nuclear/cytosol fractionation kit (Invitrogen), the cells were isolated. Briefly, 10⁶ cells were mixed with 300 μL cell disruption buffer, incubated on ice for 10 min, and centrifuged for 3 min. The supernatant was cytosol, and the precipitate was the nuclei. The supernatant was aspirated into RNase-free tubes and placed on ice to perform RNA extraction immediately. Subsequently, lncRNA GAS5 in the nucleus and cytoplasm was detected using RT-qPCR.

Tan S., Yan F., Li Q., Liang Y., Yu J., Li Z., He F., Li R, & Li M. (2020). Chlorogenic Acid Promotes Autophagy and Alleviates Salmonella Typhimurium Infection Through the lncRNAGAS5/miR-23a/PTEN Axis and the p38 MAPK Pathway. Frontiers in Cell and Developmental Biology, 8, 552020.

+ Open protocol

+ Expand

Lung Protein Expression Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Lung homogenate proteins were isolated using a Nuclear/Cytosol fractionation kit (Thermo Fisher Scientific Inc, Bartlesville, OK, USA). Equal amounts of lysate protein were separated by 8–15% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% skim milk. They were then incubated with primary antibodies against Proliferating cell nuclear antigen (PCNA), Glucose transporter 1 (GLUT1), (Santa Cruz Biotechnology, Inc., TX, USA.), Carbonic anhydrase 9 (CA9) (GeneTex, Inc. CA, USA.), c-Myc, cyclin D1, cyclin-dependent kinase 4 (CDK4), survivin, p21, HIF-1α, VEGF, Bcl2, mammalian target of rapamycin (mTOR), ERK1/2, β-catenin, the nuclear factor erythroid 2-related factor 2 (Nrf2), or β-actin (Cell Signaling Technology, Inc., Danvers, MA, USA) at a 1:1,000 dilution at 4 °C overnight. Target proteins were detected using the enhanced chemiluminescence method (Bio-Rad, Hercules, CA, USA). Signal detection was performed using ImageQuant LAS 500 Imaging System (GE Healthcare, Life Sciences, Korea). Collagenolytic activities in serum were measured by gelatin zymography. The gels were stained with Coomassie Brilliant Blue R-250 and then destained with methanol-acetic acid-water.

Kang H.S., Kwon H.Y., Kim I.K., Ban W.H., Kim S.W., Kang H.H., Yeo C.D, & Lee S.H. (2020). Intermittent hypoxia exacerbates tumor progression in a mouse model of lung cancer. Scientific Reports, 10, 1854.

+ Open protocol

+ Expand

Evaluating NF-κB Activation After Menthol Exposure

Check if the same lab product or an alternative is used in the 5 most similar protocols

24 h after menthol aerosol exposure in the VITROCELL® cloud chamber, cells in transwells were lysed using RIPA buffer. To evaluate NF-κB activation, nuclear and cytoplasmic fractions of cell suspensions were isolated after treatment using the Nuclear/Cytosol Fractionation Kit (Thermo Fischer Scientific). Western blotting was performed as previously described (Nair et al., 2014 (link)). Lysates were vortexed every 15 min for 1 h, centrifuged at 10,000 × g for 10 min at 4°C, quantified using the Pierce BCA assay kit (Thermo Scientific, Waltham, Massachusetts). Lysates were mixed with Laemmli buffer (1:4), 20 μg of protein/gel lane were separated using SDS gel electrophoresis (100 V for 2 h), and then transferred to a PVDF membrane (BioRad, Carlsbad, CA) by wet electroblotting. The membrane was blocked (5% milk in TBST buffer x 45 min) and incubated overnight at 4°C with antibodies against TRPM8 (Abcam, Cambridge, MA), SOD2 (Cell Signaling Technology, Danvers, MA), or β-actin (Cell Signaling Technology, Danvers, MA). Membranes were washed for 30 min in TBST, incubated in secondary antibody for 2 h, then developed using immunoblot reagent (Bio ad, Hercules, California ) in a ChemiDoc™ Imaging ystems (Bio ad, Hercules, California ).

Nair V., Tran M., Behar R.Z., Zhai S., Cui X., Phandthong R., Wang Y., Pan S., Luo W., Pankow J.F., Volz D.C, & Talbot P. (2020). Menthol in Electronic Cigarettes: A Contributor to Respiratory Disease?. Toxicology and applied pharmacology, 407, 115238.

+ Open protocol

+ Expand

Western Blot Analysis of API5 Localization

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

A total of 5 × 10⁵ cells were used as described previously [18 (link), 19 (link)]. Equal amounts of nuclear protein and cytosol protein were solubilized in Laemmli buffer (62.5 mM Tris/HCL pH 6.8, 10% glycerol, 2% SDS, 5% mercaptoethanol and 0.00625% bromophenol blue), boiled for 5 min, and then separated by 12% polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were probed with primary antibodies of API5 (Sigma-Aldrich, St. Louis, MO; clone# 1C2, 1:250) or GAPDH (Chemicon International, Temecula, CA; clone no. 6C5, 1:5000) in Tris-buffered saline (TBS)-T containing 5% BSA (Sigma-Aldrich) at 4°C overnight, followed by 3 washes in TBST, 5 min per wash. The membranes were incubated with the appropriate secondary antibodies for 1 hr at room temperature. Immunoreactive bands were visualized by an enhanced chemiluminescence reaction (ECL, Elpis Biotech, Daejeon, Korea). To observe the cellular localization of API5, HeLa cells were subjected to fractionation using a commercial kit (Nuclear/Cytosol Fractionation Kit, Thermo scientific, Rockford, IL) according to the manufacturer's instructions.

Cho H., Chung J.Y., Song K.H., Noh K.H., Kim B.W., Chung E.J., Ylaya K., Kim J.H., Kim T.W., Hewitt S.M, & Kim J.H. (2014). Apoptosis inhibitor-5 overexpression is associated with tumor progression and poor prognosis in patients with cervical cancer. BMC Cancer, 14, 545.

+ Open protocol

+ Expand

Western Blot Protein Quantification

Check if the same lab product or an alternative is used in the 5 most similar protocols

HBVSMCs or cerebrovascular tissues were lysed using RIPA buffer (Beyotime) containing 1% protease and phosphatase inhibitors (Merck). Nuclear proteins were extracted using a Nuclear/Cytosol Fractionation Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The protein concentration of each sample was determined using a bicinchoninic acid kit (BioRad). Equal amounts of protein were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on 8–10% gels and then transferred onto nitrocellulose membranes (Millipore). The membranes were blocked with 5% non-fat milk powder in TBST consisting of 10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20 (pH 7.6) and incubated with the appropriate antibodies at 4 °C overnight. Afterwards, the blots were visualized with HRP-conjugated secondary antibodies followed detection with a chemiluminescence system (Thermo Fisher Scientific). Image quantification was performed using ImageJ software.

Lu J., Xu F., Zhang Y., Lu H, & Zhang J. (2018). ClC-2 knockdown prevents cerebrovascular remodeling via inhibition of the Wnt/β-catenin signaling pathway. Cellular & Molecular Biology Letters, 23, 29.

+ Open protocol

+ Expand

Murine Neutrophil Protein Phosphorylation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein expression and phosphorylation of target proteins were analyzed by western blotting. Murine neutrophils were stimulated at an MOI of 1:1 with fungal particles. Following incubation for 30 min at 37°C, cells were lysed with RIPA buffer (Sigma, St. Louis, MO, USA) or a nuclear/cytosol fractionation kit (ThermoFisher Scientific, Waltham, MA, USA). All lysis buffers contain final 2.5mM Diisopropyl fluorophosphate (DFP) (Sigma, St. Louis, MO, USA), 1x protease inhibitor cocktail (Roche, Basel, Switzerland) and 1x phosphatase inhibitor cocktail (ThermoFisher Scientific, Waltham, MA, USA). A total of 40 μg lysate was loaded on 4–12% SDS-PAGE gels (Invitrogen) and transferred to 0.2 μm PVDF membranes (Bio-Rad, Hercules, CA, USA). Protein lysates were equally loaded, and expression of each target protein and its phosphorylated form was measured by GelDoc imaging system (Bio-Rad, Hercules, CA, USA) and analyzed in ImageJ (NIH). Results are expressed in arbitrary units (a.u), which is a relative number for phospho-form of the target protein compared to the total expression of the target protein, normalizing using the unstimulated WT negative control as 1.

Yoo D.G., Paracatu L.C., Xu E., Lin X, & Dinauer M.C. (2021). NADPH oxidase limits collaborative PRR signaling to regulate neutrophil cytokine production in response to fungal PAMPs. Journal of immunology (Baltimore, Md. : 1950), 207(3), 923-937.

+ Open protocol

+ Expand

Quantifying lncRNA MEG3 Subcellular Localization

Check if the same lab product or an alternative is used in the 5 most similar protocols

A nuclear/cytosol fractionation kit (Thermo Fisher Scientific) was used in this assay. The RNA level of lncRNA MEG3 in the cytoplasm and nucleus was assessed by RT-qPCR with GAPDH as a positive control for cytoplasmic RNA and U6 for nuclear RNA.

Wang L., Yu Q., Xiao J., Chen Q., Fang M, & Zhao H. (2024). Cigarette Smoke Extract-Treated Mouse Airway Epithelial Cells-Derived Exosomal LncRNA MEG3 Promotes M1 Macrophage Polarization and Pyroptosis in Chronic Obstructive Pulmonary Disease by Upregulating TREM-1 via m6A Methylation. Immune Network, 24(2), e3.

+ Open protocol

+ Expand

Western Blot Analysis of Lung Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Lung homogenate proteins were isolated using a Nuclear/Cytosol Fractionation Kit (Thermo Fisher Scientific, Inc., Bartlesville, OK, USA). Protein concentrations were determined by performing bicinchoninic acid assays. Equal amounts of proteins were separated by 10%–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% skim milk, the membrane was incubated overnight at 4°C with primary antibodies against nuclear factor-κB (NF-κB), the inhibitor of NF-κB, Kelch-like ECH-associated protein 1 (Keap1), nuclear factorerythroid-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), β-actin, and histone H3 (Cell Signaling Technology, Inc., Danvers, MA, USA). After incubation with a secondary antibody for 2 hours, target proteins were detected using the enhanced chemiluminescence method (Bio-Rad, Hercules, CA, USA). Signals were detected using the ImageQuant LAS 500 Imaging System (GE Healthcare Life Sciences, Seoul, Korea).

Kang H.H., Kim I.K., Yeo C.D., Kim S.W., Lee H.Y., Im J.H., Kwon H.Y, & Lee S.H. (2020). The Effects of Chronic Intermittent Hypoxia in Bleomycin-Induced Lung Injury on Pulmonary Fibrosis via Regulating the NF-κB/Nrf2 Signaling Pathway. Tuberculosis and Respiratory Diseases, 83(Supple 1), S63-S74.

+ Open protocol

+ Expand

Western Blot Protein Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total cellular protein in cell lysates and proteins in the prestained Protein Ladder (Thermo Fisher Scientific) were separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes. After blocking with 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature, the membranes were incubated with different primary antibodies. For details, see Supplementary Table S1. Next, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies. The Nuclear/Cytosol Fractionation Kit (Thermo Fisher, USA) was used according to the manufacturer’s protocol. In some instances, during the extraction of cytoplasmic and nuclear proteins, the proteins were visualized using electrochemiluminescence (ECL) western blotting reagent (Thermo Pierce).

Luo F., Li H., Ma W., Cao J., Chen Q., Lu F., Qiu M., Zhou P., Xia Z., Zeng K., Zhan J., Zhou T., Luo Q., Pan W., Zhang L., Lin C., Huang Y., Zhang L., Yang D, & Zhao H. (2023). The BCL-2 inhibitor APG-2575 resets tumor-associated macrophages toward the M1 phenotype, promoting a favorable response to anti-PD-1 therapy via NLRP3 activation. Cellular and Molecular Immunology, 21(1), 60-79.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!