Ne per nuclear cytoplasmic extraction reagent kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The NE-PER Nuclear Cytoplasmic Extraction Reagent kit is a laboratory product designed to extract and separate nuclear and cytoplasmic proteins from mammalian cells. The kit contains pre-formulated reagents to facilitate the extraction process.

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28 protocols using ne per nuclear cytoplasmic extraction reagent kit

Nuclear Extraction of Cardiac Fibroblasts

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The nuclear extraction was prepared using an NE-PER nuclear cytoplasmic extraction reagent kit (Cat# 78833, Thermo Fisher Scientific) according to the manufacturer’s instruction. Briefly, cardiac fibroblasts from Mcpt4^−/− and WT mice were suspended in 200 μl of cytoplasmic extraction reagent-I and incubated on ice for 10 min followed by addition of 11 μl of a second cytoplasmic extraction reagent-II. After centrifugation, the supernatant fraction (cytoplasmic extract) was transferred to a pre-chilled tube. The insoluble pellet fraction, which contains crude nuclei, was re-suspended in 100 μl of a nuclear extraction reagent. After centrifugation, the resulting supernatant, constituting the nuclear extract, was used for the western blot to detect p-Smad2, p-Smad3, GAPDH, and histone-H3.

Wang Y., Liu C.L., Fang W., Zhang X., Yang C., Li J., Liu J., Sukhova G.K., Gurish M.F., Libby P., Shi G.P, & Zhang J. (2019). Deficiency of mouse mast cell protease 4 mitigates cardiac dysfunctions in mice after myocardium infarction. Biochimica et biophysica acta. Molecular basis of disease, 1865(6), 1170-1181.

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Cellular Fractionation of A549 Cells

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A549 cells were transfected with scramble or CDK16 siRNAs. After 48 h, the cells were collected. Cytoplasmic and nuclear fractions were separated using NE-PER Nuclear Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific) according to the manufacturer's procedures.

Xie J., Li Y., Jiang K., Hu K., Zhang S., Dong X., Dai X., Liu L., Zhang T., Yang K., Huang K., Chen J., Shi S., Zhang Y., Wu G, & Xu S. (2018). CDK16 Phosphorylates and Degrades p53 to Promote Radioresistance and Predicts Prognosis in Lung Cancer. Theranostics, 8(3), 650-662.

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Nuclear Cytoplasmic Extraction for RNA Analysis

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The NE-PER Nuclear Cytoplasmic Extraction Reagent kit (Thermo Scientific, USA) was used for nucleocytoplasmic separation. RAW264.7 cells were washed and then placed in a 1.5 mL EP tube and centrifuged at 500× g. Precooled 500 μL cytoplasmic extraction reagent I was then added to the cell precipitate and vortexed for 15 s to suspend the cell precipitate. This was placed on ice for 10 min. Then, 11 mL of a second cytoplasmic extraction reagent II was added to the sample. It was then placed on ice and centrifuged for 5 min at 16,000× g. The supernatant was placed into a new precooled EP tube. Precooled 250 μL NER (Nuclear Extraction Agent) was used to resuspend the sediment (which contained crude nuclei), and then the sample was incubated on ice after vortexing for 15 s. After centrifuging at 4 °C for 10 min, the supernatant (nuclear extract) was transferred into a new precooled EP tube. The RNA of the nuclear extract and cytoplasmic extract were extracted, and the expression of Gm16685 in the nucleus and cytoplasm was further analyzed using qRT-PCR.

Zhao R., Tang X., Lin H., Xing C., Xu N., Dai B., Wang P., Shao W., Liu M., Shen J., Deng S, & Ren C. (2023). Knocking Down Gm16685 Decreases Liver Granuloma in Murine Schistosomiasis Japonica. Microorganisms, 11(3), 796.

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Cytoplasmic and Nuclear Fractionation

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Cells were transfected with scramble or SDH5-siRNA. After 48 h, the cells were collected. Cytoplasmic and nuclear fractions were separated using an NE-PER Nuclear Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific) according to the manufacturer's procedures.

Zong Y., Li Q., Zhang F., Xian X., Wang S., Xia J., Li J., Tuo Z., Xiao G., Liu L., Li G., Zhang S., Wu G, & Liu J. (2019). SDH5 Depletion Enhances Radiosensitivity by Regulating p53: A New Method for Noninvasive Prediction of Radiotherapy Response. Theranostics, 9(22), 6380-6395.

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Nuclear and Cytoplasmic Fractionation

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Nuclear and cytoplasmic fractions were prepared using the NE-PER Nuclear Cytoplasmic Extraction Reagent kit from ThermoFisher scientific (Waltham, MA, USA) according to the manufacturer’s instruction.

Zare A., Petrova A., Agoumi M., Amstrong H., Bigras G., Tonkin K., Wine E, & Baksh S. (2018). RIPK2: New Elements in Modulating Inflammatory Breast Cancer Pathogenesis. Cancers, 10(6), 184.

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Nuclear Protein Extraction and Western Blot

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According to the manufacturer’s instructions, nuclear or cytoplasmic proteins from the cells were isolated using a NE-PER Nuclear Cytoplasmic Extraction Reagent Kit (Thermo Fisher Scientific, Waltham, USA). Nuclear protein was separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Schleicher & Schuell Bioscience, Inc., Keene, NH, USA). Western blot analysis was performed using anti-SMARCA4 (Abcam, Cambridge, UK; ab110641) and anti-histone H3 (Abcam, Cambridge, UK; ab1791) antibodies. Histone H3 protein was used as an internal control.

Choi H.I., An G.Y., Baek M., Yoo E., Chai J.C., Lee Y.S., Jung K.H, & Chai Y.G. (2021). BET inhibitor suppresses migration of human hepatocellular carcinoma by inhibiting SMARCA4. Scientific Reports, 11, 11799.

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Nuclear Extraction Protocol

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Nuclear extraction was performed using an NE‐PER Nuclear Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific) according to the manufacturer’s instruction.

Yi Z., Deng M., Scott M.J., Fu G., Loughran P.A., Lei Z., Li S., Sun P., Yang C., Li W., Xu H., Huang F, & Billiar T.R. (2020). Immune‐Responsive Gene 1/Itaconate Activates Nuclear Factor Erythroid 2–Related Factor 2 in Hepatocytes to Protect Against Liver Ischemia–Reperfusion Injury. Hepatology (Baltimore, Md.), 72(4), 1394-1411.

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Evaluating Apoptotic Signaling in Chemoresistant Lung Cancer Cells

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Based on Lee et al. [25 (link)], Western blotting was conducted in A549/CR and H460/CR cells exposed to PGG (0, 6.25, 12.5 and 25 μM) for 48 h. In brief, whole cell lysates were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail, and isolated proteins in the supernatants were quantified and electrotransferred onto a Hybond enhanced chemiluminescence (ECL) transfer membrane. The membranes were probed with primary antibodies for PTEN, p-AKT, PARP, cellular inhibitor of apoptosis 1 (c-IAP1), c-IAP2, Survivin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), Caspase-8, -9, -7, Cleaved caspase-3, BAX, Bcl-2, Bcl-xL, p-ATR, p-Chk2, p-BRCA-1, p-H₂AX (Cell signaling Technology, Danvers, MA, USA), p53 (Oncogene Research Products, San Diego, CA, USA), XIAP (Becton Dickinson and Company BD Biosciences, San Jose, CA, USA), β-actin (Sigma Aldrich, St Louis, MO) and horseradish peroxidase-conjugated secondary antibody. Additionally, nuclear extract was isolated for DNA damage proteins using an NE-PER Nuclear Cytoplasmic Extraction Reagent kit (Thermo Scientific, Rochester, NY, USA).

Kim J.H., Im E., Lee J., Lee H.J., Sim D.Y., Park J.E., Ahn C.H., Kwon H.H., Shim B.S., Kim B, & Kim S.H. (2022). Apoptotic and DNA Damage Effect of 1,2,3,4,6-Penta-O-galloyl-beta-D-glucose in Cisplatin-Resistant Non-Small Lung Cancer Cells via Phosphorylation of H2AX, CHK2 and p53. Cells, 11(8), 1343.

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Nuclear Extraction Protocol for Cell Fractionation

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Nuclear extraction was conducted using an NE- PER Nuclear Cytoplasmic Extraction Reagent kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. In brief, the transfected HepG2 cell pellets were suspended in cytoplasmic extraction reagent I by vortexing and incubated on ice for 10 min; then, a second cytoplasmic extraction reagent II was added. After the cells were centrifuged, the supernatant fraction (cytoplasmic extract) was transferred to a prechilled tube. The insoluble pellet fraction containing crude nuclei was resuspended in a nuclear extraction reagent. The final supernatant, constituting the nuclear extract, was used for the subsequent experiments.

Kim J.H., Jung J.H., Lee H.J., Sim D.Y., Im E., Park J., Park W.Y., Ahn C.H., Shim B.S., Kim B, & Kim S.H. (2021). UBE2M Drives Hepatocellular Cancer Progression as a p53 Negative Regulator by Binding to MDM2 and Ribosomal Protein L11. Cancers, 13(19), 4901.

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Protein Extraction and Western Blot Analysis

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Total protein was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime, China). An NE-PER™ Nuclear Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific, MA) was used according to the manufacturer’s instructions for extracting nuclear and cytoplasmic proteins from the different groups.
A 12% sodium dodecyl sulfate‐polyacrylamide gel was chosen for total protein separation, and the proteins were then transferred to nitrocellulose membranes (Millipore, USA). The membranes were incubated with primary antibodies, including anti-LC3B (Abcam Cat# ab192890, RRID: AB_2827794), anti-P62/SQSTM1 (Abcam Cat# ab207305, RRID: AB_2885112), anti-β-actin (Proteintech# 20536-1-AP), anti-LDHA (Cell Signaling Technology Cat# 3582, RRID: AB_2066887), anti-β-catenin (Cell Signaling Technology Cat# 8480), anti-p-β-catenin (Cell Signaling Technology Cat# 4176), anti-c-Myc (Covance Cat# MMS-150P-1000, RRID: AB_291322), anti-GSK3-β (Cell Signaling Technology Cat# 121456), anti-Axin1 (Cell Signaling Technology Cat# 2087, RRID: AB_2274550) and anti-Histone H3 (Cell Signaling Technology Cat# 4499, RRID: AB_10544537). Enhanced chemiluminescence reagents (Millipore, USA) were used to assess protein expression.

Li T., Tong H., Yin H., Luo Y., Zhu J., Qin Z., Yin S, & He W. (2021). Starvation induced autophagy promotes the progression of bladder cancer by LDHA mediated metabolic reprogramming. Cancer Cell International, 21, 597.

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