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Nxtract cellytic nuclear extraction kit

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The NXTRACT CelLytic Nuclear Extraction Kit is a laboratory product designed for the isolation and extraction of nuclear proteins from eukaryotic cells. It provides a standardized and efficient method for the separation of nuclear and cytoplasmic fractions.

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Lab products found in correlation

Hybond, by Cytiva (4 mentions) 2 mercaptoethanol, by Bio-Rad (2 mentions) Irdye 680 rabbit anti mouse secondary antibody, by LI COR (2 mentions) Hybond ecl, by Cytiva (2 mentions) Id image analysis software, by Kodak (2 mentions) Hybond c extra, by GE Healthcare (2 mentions) Readyprep protein extraction kit, by Bio-Rad (2 mentions) Phospho creb, by Cell Signaling Technology (1 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions) Egr 1, by Santa Cruz Biotechnology (1 mentions) Protein assay kit, by Bio-Rad (1 mentions) Bromophenol blue, by Merck Group (1 mentions) Gapdh, by Abcam (1 mentions) Glycerol, by Merck Group (1 mentions) Tris hcl, by Merck Group (1 mentions) 2 mercaptoethanol, by Merck Group (1 mentions) Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (1 mentions) Quantity one 1 d analysis software, by Bio-Rad (1 mentions)

Spelling variants of this product

CelLytic NuCLEAR Extraction Kit (141 citations) Nuclear Extraction Kit (104 citations) NXTRACT (5 citations) NXTRACT NuCLEAR kit (1 citations)

9 protocols using nxtract cellytic nuclear extraction kit

1

Nuclear Protein Extraction and Western Blotting

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Individual wells from cultured cells and tissue from mice were collected as described above. The homogenate was then fractionated with a NXTRACT CelLytic™ NuCLEAR™ Extraction Kit (Sigma) according to the manufacturer’s instructions. Following this, samples were prepared on ice (to a final volume of 40 microliters) in a mixture of Laemmli sample buffer and 2-mercaptoethanol (BioRad) and then vortexed and denatured for 10 min at 70°C. Gels were run and proteins transferred onto nitrocellulose membrane (Hybond-ECL, Amersham). The membrane was blocked with Odyssey Blocking Buffer (LiCore P/N 927–40100) for 1 h at room temperature, washed with Trizma-buffered saline with 1% Triton-X-100 (TBST) for 5 min (3x) and incubated with 5ml of mouse monoclonal antibody (Adar1 1:1000; Santa Cruz sc-73408 B) in blocking buffer for 24 hours at 4°C. The membrane was washed with TBST (3x), incubated for 1 h with IRDye 680 rabbit anti-mouse secondary antibody (1:10000; LI-COR) in TBST, and washed in TBST for 10 min (3x). Optical density readings of the blots were made using the LI-COR analysis system.
Marshall P.R., Zhao Q., Li X., Wei W., Periyakaruppiah A., Zajaczkowski E.L., Leighton L.J., Madugalle S.U., Basic D., Wang Z., Yin J., Liau W.S., Gupte A., Walkley C.R, & Bredy T.W. (2020). Dynamic regulation of Z-DNA in the mouse prefrontal cortex by the RNA editing enzyme Adar1 is required for fear extinction. Nature neuroscience, 23(6), 718-729.
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2

Western Blot Analysis of Fetal Heart Proteins

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Fetal hearts and left ventricles of offspring hearts were homogenized in RIPA lysis buffer (Life Technologies, Carlsbad, CA) supplemented with Halt protease inhibitors cocktail (Pierce Biotechnology, Rockford, IL). Nuclear extraction was prepared using NXTRACT CelLytic Nuclear Extraction Kit (Sigma, St. Louis, MO), following the manufacturer's instructions. Protein concentrations were determined using the BCA assay kit (Pierce). Samples with equal amounts of proteins were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in TBS for 1 hour at room temperature and then probed with primary antibodies against GR, Egr-1 (Santa Cruz Biotechnology, Santa Cruz, CA), Sp1 (Active Motif, Carlsbad, CA), CREB and phospho-CREB (Cell Signaling, Danvers, MA). After a brief wash, membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. Protein signal was visualized with enhanced chemiluminescence reagents (Pierce) and blots were exposed to Hyperfilm. The results were quantified with the Kodak electrophoresis documentation and analysis system and Kodak ID image analysis software. The target protein abundance was normalized to the abundance of β-actin.
Xiong F., Lin T., Song M., Ma Q., Martinez S.R., Lv J., MataGreenwood E., Xiao D., Xu Z, & Zhang L. (2016). Antenatal Hypoxia Induces Epigenetic Repression of Glucocorticoid Receptor and Promotes Ischemic-Sensitive Phenotype in the Developing Heart. Journal of molecular and cellular cardiology, 91, 160-171.
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3

Measuring Nuclear HAT Activity

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Nuclei were isolated from PC3 cells 1 hour post-radiation using the NXTRACT CelLytic NuCLEAR Extraction Kit (Sigma, St. Louis, MO). Protein concentrations of the nuclear extracts were determined by the Bradford method. Equal amounts of nuclear extract protein (~5 μg) were analyzed for HAT activity using the EpiQuick Activity/Inhibition Assay Kit (Epigetek, Farmingdale, NY) according to manufacturer’s suggestions.
Tong Q., Weaver M.R., Kosmacek E.A., O’Connor B., Harmacek L., Venkataraman S, & Oberley-Deegan R.E. (2016). MnTE-2-PyP reduces prostate cancer growth and metastasis by suppressing p300 activity and p300/HIF-1/CREB binding to the promoter region of the PAI-1 gene. Free radical biology & medicine, 94, 185-194.
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4

Quantifying Nuclear Protein Sp1 Levels

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Nuclear extracts were prepared from uterine arteries using NXTRACT CelLytic Nuclear Extraction Kit (Sigma, St Louis, MO). Protein concentrations were measured using a protein assay kit (Bio-Rad, Hercules, CA). Samples with equal amounts of protein were loaded onto 7.5% polyacrylamide gel with 0.1% SDS and separated by electrophoresis at 100 V for 90 minutes. Proteins were then transferred onto nitrocellulose membranes. Nonspecific binding sites was blocked for 1 hour at room temperature in a Tris-buffered saline solution containing 5% dry-milk. The membranes were then probed with primary antibody against Sp1 (Active motif; Carlsbad, CA). After washing, membranes were incubated with secondary horseradish peroxidase-conjugated antibodies. Proteins were visualized with enhanced chemiluminescence reagents, and blots were exposed to Hyperfilm. Results were analyzed with the Kodak ID image analysis software.
Chen M., Dasgupta C., Xiong F, & Zhang L. (2014). Epigenetic Up-regulation of Large-Conductance Ca2+-Activated K+ Channel Expression in Uterine Vascular Adaptation to Pregnancy. Hypertension, 64(3), 610-618.
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5

Nuclear Protein Extraction and Western Blotting

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Individual wells from cultured cells and tissue from mice were collected as described above. The homogenate was then fractionated with a NXTRACT CelLytic™ NuCLEAR™ Extraction Kit (Sigma) according to the manufacturer’s instructions. Following this, samples were prepared on ice (to a final volume of 40 microliters) in a mixture of Laemmli sample buffer and 2-mercaptoethanol (BioRad) and then vortexed and denatured for 10 min at 70°C. Gels were run and proteins transferred onto nitrocellulose membrane (Hybond-ECL, Amersham). The membrane was blocked with Odyssey Blocking Buffer (LiCore P/N 927–40100) for 1 h at room temperature, washed with Trizma-buffered saline with 1% Triton-X-100 (TBST) for 5 min (3x) and incubated with 5ml of mouse monoclonal antibody (Adar1 1:1000; Santa Cruz sc-73408 B) in blocking buffer for 24 hours at 4°C. The membrane was washed with TBST (3x), incubated for 1 h with IRDye 680 rabbit anti-mouse secondary antibody (1:10000; LI-COR) in TBST, and washed in TBST for 10 min (3x). Optical density readings of the blots were made using the LI-COR analysis system.
Marshall P.R., Zhao Q., Li X., Wei W., Periyakaruppiah A., Zajaczkowski E.L., Leighton L.J., Madugalle S.U., Basic D., Wang Z., Yin J., Liau W.S., Gupte A., Walkley C.R, & Bredy T.W. (2020). Dynamic regulation of Z-DNA in the mouse prefrontal cortex by the RNA editing enzyme Adar1 is required for fear extinction. Nature neuroscience, 23(6), 718-729.
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6

Quantifying CREB Activity in Irradiated Cells

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Nuclei were isolated from PC3 cells 1–24 hours post-radiation using the NXTRACT CelLytic NuCLEAR Extraction Kit (Sigma). Protein concentrations of the nuclear extracts were determined by the Bradford method. Equal amounts of nuclear extract protein (~5 μg) were analyzed for CREB activity using the TransAM CREB Transcription Factor Assay Kits (Active Motif) according to manufacturer’s suggestions.
Tong Q., Weaver M.R., Kosmacek E.A., O’Connor B., Harmacek L., Venkataraman S, & Oberley-Deegan R.E. (2016). MnTE-2-PyP reduces prostate cancer growth and metastasis by suppressing p300 activity and p300/HIF-1/CREB binding to the promoter region of the PAI-1 gene. Free radical biology & medicine, 94, 185-194.
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7

Western Blot Analysis of YAP

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Constitutive proteins were extracted from cell lysates using the ReadyPrep Protein Extraction Kit (Bio-Rad). Cell nuclear extracts were isolated using the NXtract CelLytic NuCLEAR Extraction Kit (Sigma-Aldrich). Protein concentration was measured by Coomassie Blue-G Dye-binding method. Also, 100 μg of proteins were resuspended in sample buffer (1:1) consisting of 4% SDS (Sigma-Aldrich), 10% 2-mercaptoethanol (Sigma-Aldrich), 20% glycerol (Sigma-Aldrich), 0.004% bromophenol blue (Sigma-Aldrich), and 0.125 M Tris–HCl (Sigma-Aldrich) at pH 6.8. Equal amounts of proteins were loaded, electrophoresed on SDS–polyacrylamide gels, transferred onto 0.45 μm pore size nitrocellulose membranes (Hybond-C Extra, GE Healthcare Life Sciences), and probed with YAP primary antibody (1:1000, Cell signaling, 14,074). Protein bands were visualized by the WesternBreeze chemiluminescent kit, and densitometric analysis was performed using the ImageJ software (ImageJ software version 1.53j). GAPDH (1:1000, Abcam, ab8245) was used as loading control for protein normalization.
Pennarossa G., Arcuri S., De Iorio T., Ledda S., Gandolfi F, & Brevini T.A. (2023). Combination of epigenetic erasing and mechanical cues to generate human epiBlastoids from adult dermal fibroblasts. Journal of Assisted Reproduction and Genetics, 40(5), 1015-1027.
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8

EMSA Analysis of miR-133a Promoter

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Nuclear extract (NE) was prepared from fetal rat hearts using NXTRACT CelLytic Nuclear Extraction Kit (Sigma). The oligonucleotide probes containing HBS (sense: 5′-ctgggaaatgtgagtggaaacatgag; anti-sense: 5′-ctcatgtttccactcacatttcccag) and HAS (sense: 5′-gaaacatgagacacgtctttatttggca; anti-sense: 5′-tgccaaataaagacgtgtctcatgtttc) motifs in the miR-133a promoter were biotin-labeled and then subjected to EMSA using LightShift Chemiluminescent EMSA Kit (Pierce Biotechnology). For cold competition (CC), 200-fold molar excess of unlabeled homologous oligonucleotides were added to EMSA binding reactions. As described earlier [63 (link)], for super-shift assays, 1 μl of normal rabbit serum or HIF-1α antiserum (Active Motif) was pre-incubated with NE in the 1x binding buffer for 2 hours at 4°C. This was followed by addition of biotinylated probe and poly (dI-dC). The mixture was separated by electrophoresis and transferred to a Biodyne nylon membrane (Pierce). Membranes were blocked and the shifted or super-shifted bands were visualized using the reagents provided in the LightShift kit (Pierce) following Pierce protocol and recorded on an x-ray film.
Shin A.N., Han L., Dasgupta C., Huang L., Yang S, & Zhang L. (2018). SIRT1 increases cardiomyocyte binucleation in the heart development. Oncotarget, 9(8), 7996-8010.
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9

Protein Extraction and Western Blot Analysis

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Cells were lysed and constitutive proteins were extracted using a ReadyPrep Protein Extraction Kit (Bio-Rad). Nuclear extracts from the cells were isolated using the NXtract CelLytic NuCLEAR Extraction Kit (Sigma). Protein concentration was assessed by Coomassie Blue-G Dye-binding method. 100 μg of proteins were resuspended in sample buffer (1:1) consisting of 4% (wt/vol) SDS, 10% 2-mercaptoethanol, 20% (wt/vol) glycerol, 0.004% bromophenol blue, and 0.125 M Tris-HCl at pH 6.8. Equal amounts of total protein were loaded and electrophoresed on a SDS-polyacrylamide gels. Proteins were then transferred onto 0.45 μm pore size nitrocellulose membranes (Hybond-C Extra, GE Healthcare Life Sciences) and probed with primary antibodies listed in Table 2. Protein bands were visualized by the WesternBreeze chemiluminescent kit. Densitometric analysis was performed with Quantity One 1-D analysis software (Bio-Rad).
Pennarossa G., Manzoni E.F.M., Ledda S., deEguileor M., Gandolfi F, & Brevini T.A.L. (2019). Use of a PTFE Micro-Bioreactor to Promote 3D Cell Rearrangement and Maintain High Plasticity in Epigenetically Erased Fibroblasts. Stem cell reviews and reports, 15(1).
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