SAHA was synthesized by Prof. J. P Liou in Taipei Medical University [24 (link)] and was dissolved in dimethylsulfoxide (DMSO) (Fisher Scientific, 67-68-5). SB203580, a specific p38 inhibitor, was purchased from Selleckchem (S1076) and dissolved in DMSO. The following commercial antibodies were used in Western blotting and Immunofluorescence: CD133 (Proteintech, 18470-1-AP), Bmi1 (GeneTex, GTX114008), Nanog (GeneTex, GTX100863), Oct3/4 (Santa Cruz, SC5279), Vimentin (GeneTex, GTX100619), Sox2 (GeneTex, GTX101507), tubulin (Proteintech, 66031-1-lg), PARP (Cell Signaling, 9542S), caspase-8 (GeneTex, GTX110723), caspase-9 (Cell Signaling, 9502S), caspase-3 (Cell Signaling, 9661S), cyclin B1 (Santa Cruz, SC245), CDK1 (Santa Cruz, SC8395), p21 (Cell Signaling, 2947S), PCNA (Cell Signaling, 2586S), Phospho-p38 (p-p38, T180/Y182) (Cell Signaling, 9211S), p38 (Cell Singling, 8690S), p53 (Cell Signaling, 2524S), Phospho-p53 (Ser15) (Cell Signaling, 9284S), Phospho-p53 (Ser33) (Cell Signaling, 2526S), and Alexa Fluor 594-conjugated goat anti-rabbit IgG polyclonal antibodies (Invitrogen, A-11037).
Nanog
Manufactured by GeneTex
Sourced in United States
Nanog is a laboratory reagent used for molecular biology research. It is a protein that functions as a transcription factor, playing a critical role in the self-renewal of undifferentiated embryonic stem cells. Nanog is a key regulator of pluripotency, the ability of stem cells to differentiate into various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Vimentin, by GeneTex (2 mentions)
Gapdh, by Merck Group (2 mentions)
Cd133, by Proteintech (1 mentions)
Caspase 8, by GeneTex (1 mentions)
Oct3 4, by Santa Cruz Biotechnology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Phospho p53 ser33, by Cell Signaling Technology (1 mentions)
Cyclin b1, by Santa Cruz Biotechnology (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
Phospho p53 ser15, by Cell Signaling Technology (1 mentions)
Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (1 mentions)
Sb203580, by Selleck Chemicals (1 mentions)
Tra 1 60, by R&D Systems (1 mentions)
Ssea4, by STEMCELL (1 mentions)
Facstar flow cytometer, by BD (1 mentions)
Ssea 4, by Merck Group (1 mentions)
Ssea 3, by BD (1 mentions)
Icam 1, by GeneTex (1 mentions)
E cadherin, by GeneTex (1 mentions)
N cadherin, by GeneTex (1 mentions)
9 protocols using nanog
1
SAHA Modulates Cell Stemness and Apoptosis
2
Pluripotency Marker Immunostaining in iPSCs
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Mature iPSC colonies in a 24 well plate were stained with Tra-1-60 (10 μg/well; R&D systems), SSEA4 (6 μg/well; StemCell Technologies), and Nanog (1:200; Genetex, Irvine, CA) primary antibodies. Cells were fixed in 4% paraformaldehyde for 15 min and washed in 0.1% Triton X-100 in phosphate buffered saline (PBS-T) three times for 5 min at room temperature. Cells were simultaneously blocked and permeabilized in a solution of 50% PBS, 45% sterile-deionized water, 0.15% Triton X-100, and 5% serum (based on secondary antibody) for 1 h at room temperature. Primary antibodies were added to 200 μl of permeabilization/blocking solution per well and incubated overnight at 4 °C. After three washes (5 min each) with PBS-T, cells were incubated in 200 μl of 1% BSA in PBS with secondary antibodies (1:250) for 30 min at room temperature. Cells were then washed, incubated with DAPI (1:500) in PBS-T, washed 3 more times, and visualized using a fluorescence microscope.
3
Characterization of Pluripotent Stem Cells
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APOSCs were dissociated by 0.25% trypsin-EDTA, washed with PBS containing 2% BSA, and incubated with primary antibodies for SSEA-4 (1: 50, Millipore), SSEA-3 (PE-conjugated, BD), or ICAM-1 (1:100, GeneTex). For intracellular proteins, cells were fixed and permeabilized using a cell staining kit (eBiocience 88-8115) before applying primary antibodies, Oct-4 (1:100, Abcam), Nanog (1:100, GeneTex), and Sox-2 (1:50, Millipore). Cells were then detected by anti-mouse IgG, anti-rabbit IgG or anti-rat IgM conjugated with FITC. FITC-conjugated IgG2a antibodies were used as isotype controls. Thereafter, the cells were analyzed using a FACSTAR+ flow cytometer (Becton Dickinson).
4
Comprehensive Histone and EMT Marker Analysis
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Primary antibodies for H3K4me3 (GTX128954), H3K9me3 (GTX121677), H3K27me3 (GTX121184), H3K36me3 (GTX54109), H3K79me3 (GTX54111), histone H3 (GTX122148), MMP2 (GTX104577), MMP3 (GTX100723), MMP9 (GTX100458), N-cadherin (GTX101141), E-cadherin (GTX100433), vimentin (GTX100619), Snail (GTX100754), TWIST-1 (GTX60776), Nanog (GTX100863), OCT4 (GTX101497), SOX2 (GTX101507), ABCB1 (GTX108354), ABCG2 (GTX100437) and ABCC1 (GTX88673) were purchased from GeneTex International Corporation (Hsinchu City, Taiwan). Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase and rabbit polyclonal antibodies speci c for β-actin (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; cat. no. SI-A5441; 1:10,000) were used in this study. All other chemicals were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).
5
Quantitative Immunoblotting for Stemness Markers
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Immunoblotting analysis was performed as described in a previous article [45 (link)]. Western blot images were acquired using a chemiluminescence reagent (WBKLS0500, Merck Millipore) and then quantified using the ChemiDoc XRS + imaging system (BIO-RAD). The antibodies used were as follows: α-tubulin (GTX112141, GeneTex), RAD51 (GTX100469, GeneTex), γ-H2AX (GTX61796, GeneTex), CD44 (5640, Cell Signaling Technology), Oct-4 (GTX101497, GeneTex), SOX2 (GTX101507, GeneTex), Nanog (GTX100863, GeneTex), and CD133 (GTX100567, Genetex). HRP-conjugated goat anti-rabbit and anti-mouse antibodies were obtained from GeneTex (Irvine, CA).
6
Protein Expression Analysis by Immunoblotting
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The immunoblot procedure was performed as described previously (Wu et al. 2011) (link). Antibodies specific for cyclin E1, cyclin E2 and CDK2 (Santa Cruz Biotechnology Inc.), p21 (Thermo Fisher Scientific Inc.), p27 (Sigma-Aldrich), CD133 (Miltenyi Biotec, Auburn, CA, USA), CD44, Sox2, Nanog (GeneTex, Inc., Irvine, CA, USA) and GAPDH (Merck Millipore) were used. Band intensities were calculated using Image Gauge software (Fujifilm, Tokyo, Japan). The PVDF membranes were reprobed for different antibodies after using TOOLStripping Buffer (BIOTOOLS CO., LTD. Taiwan). The signal intensities of expression of target genes were normalized to those of GAPDH.
7
Proteomic Analysis of Cell Lysates
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For total lysate preparation, cells were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% sodium dodecyl sulfate, and 1% Nonidet P-40 (NP-40); pH 7.4) supplemented with proteinase inhibitors (complete proteinase inhibitors, Roche, Indianapolis, IN, USA). Protein lysates (30 μg per lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and processed for immunoblotting with antibodies against cleaved PARP (Enogene, Atlanta, GA, USA, E11-0365L), CD44 (GeneTex, Irvine, CA, USA, CTX102111), ALDH1 (GeneTex, GTX123973), Nanog (GeneTex, GTX100863), Oct4 (GeneTex, GTX101497), Bmi1 (GeneTex, GTX114008), Snail (Abcam, Cambridge, UK, Ab78105), Twist (GeneTex, GTX127310), FoxM1 (GeneTex, GTX100276), and Tubulin (Enogene, E1C601), respectively. Signals were detected using an enhanced chemiluminescence system (ECL, NEN Life Science, Boston, MA, USA).
8
Antibodies for Pluripotency Protein Detection
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9
Immunofluorescence Staining of Cells
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Tissue cryosections and cells were fixed with 4 % paraformaldehyde/PBS (Nacalai Tesque) for 15 min at room temperature. After 3 washes with PBS, cells were immersed in 0.2 % Triton X-100/PBS for 15 min at room temperature, followed by incubation with blocking solution (Sigma-Aldrich) consisting of 5 % normal FBS (MilliporeSigma, Burlington, MA, USA), 1 % BSA and PBS (Nacalai Tesque) for 30 min at room temperature. Cells were incubated in primary antibody solution for 30 min at room temperature, then washed 3 times with 1 % BSA/PBS, then in secondary antibody solution for 30 min at room temperature and washed 3 times with PBS. Finally, cells were observed and photographed using a confocal microscope. The following antibodies were used: NANOG (#GTX100863; GeneTex, Irvine, CA, USA), SFTPB (#PA5–42,000; Thermo Fisher Scientific), SFTPC (#AB3786; Sigma-Aldrich), HOPX (#PA5–48,205, Thermo Fisher Scientific), NKX2–1 (#MA5–31,938, Thermo Fisher Scientific), CPM (#DDX0520P-100; Novus Biologicals, Englewood, CO, USA), EPCAM (#2929; Cell Signaling Technology, Danvers, MA, USA), dsRNA (#MABE1134; Sigma-Aldrich).
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