Nanog

Manufactured by Santa Cruz Biotechnology

Sourced in United States, United Kingdom

Nanog is a protein that plays a crucial role in the maintenance of embryonic stem cell pluripotency and self-renewal. It is a transcription factor that is essential for the derivation and propagation of undifferentiated embryonic stem cells.

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Anti-Nanog (37 citations) Rabbit anti-NANOG (10 citations) Mouse anti-Nanog (6 citations) Anti-Nanog antibody (4 citations) Goat anti-Nanog (3 citations) NANOG (H-155, (2 citations) Anti-Nanog (Clone H-155 (2 citations) NANOG siRNA (2 citations) Anti-Nanog (sc-33760, (2 citations) Anti-NANOG (sc-293121, (2 citations)

81 protocols using nanog

Stem Cell Characterization by Immunostaining

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The primary antibodies were CD133-PE (1:50, Miltenyi Biotec), CD44 (1:400; Santa Cruz), CD105 (1:100; Santa Cruz), Oct4 (1:100; Santa Cruz) and Nanog (1:200; Santa Cruz). The second antibody was incubated with FITC-labeled goat anti-mouse IgG (1:200; SantaCruz). All were prepared according to the Manufacturer’s protocols. Cells were fixed with 4 % of paraformaldehyde and when needed, permeabilized with 0.05 % of Triton X-100 in PBS at room temperature for 20 min. Samples were blocked with 1 % of bovine serum albumin (Sigma) and incubated with appropriate primary antibody at 37 °C for 1 h. After washing extensively, they were incubated with second antibody at 37 °C for 1 h. After immunolabeling and washing procedure, nuclei were then counterstained with 4′6-diamidino-2-phenylindole (DAPI; Sigma). Coverslips were viewed under fluorescence microscopy (Olympus LX51, Tokyo, Japan) and photos were taken using 100-fold magnification.

Zhang Y., Sun B., Zhao X., Sun H., Cui W., Liu Z., Yao X, & Dong X. (2016). Spheres derived from the human SN12C renal cell carcinoma cell line are enriched in tumor initiating cells. Journal of Experimental & Clinical Cancer Research : CR, 35, 163.

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Antibody Characterization for Cell Signaling

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Antibodies against ADAM9, phospho-AKT (Ser473), β-actin, phospho-FAK (Tyr397), ubiquitin and phospho-GSK3b (Ser9) were purchased from Cell Signaling (Boston, MA). Antibodies against laminin-5 (γ² chain), Sox2, Oct-3/4, Nanog and E-cadherin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against fibronectin, vimentin and N-cadherin were purchased from GeneTex (San Antonio, TX). Antibodies against Col XVII, Col XVII (NC16A-3) and Snail were purchased from Abcam (Cambridge, MA). Antibodies against ADAM10 were from Millipore (Amersham Pharmacia Biotech, Piscataway, NJ). Antibodies against phospho-Snail (Ser246) were from OriGene Technologies, Inc. (Rockville, MD).

Liu C.C., Lin J.H., Hsu T.W., Hsu J.W., Chang J.W., Su K., Hsu H.S, & Hung S.C. (2016). Collagen XVII/laminin-5 activates epithelial-to-mesenchymal transition and is associated with poor prognosis in lung cancer. Oncotarget, 9(2), 1656-1672.

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Characterization of Human Embryonic Stem Cells

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Immunostaining, AP staining and karyotype analysis of haEpiSCs were performed as described previously.²⁸ Primary antibodies including TFE3 (Sigma, HPA023881, USA), H3K27me3 (AB clonal, A2363, China), OCT4 (Abcam, ab181557, UK), NANOG (Santa Cruz, sc‐374103, USA), PAX6 (Abcam, ab5790, UK), SOX1 (R&D, AF3369, USA), TUJ1 (Abcam, ab8207, UK), and SSEA‐1 (CST, 4744, USA). The fluorescent secondary antibodies and DAPI were purchased from the Abcam company (UK). Immunofluorescence images were captured with a TCS SP8 confocal laser scanning microscope (Leica, Germany).

Gao Q., Zhang W., Zhao Y., Tian Y., Wang Y., Zhang J., Geng M., Xu M., Yao C., Wang H., Li L., Liu Y, & Shuai L. (2021). High‐throughput screening in postimplantation haploid epiblast stem cells reveals Hs3st3b1 as a modulator for reprogramming. Stem Cells Translational Medicine, 10(5), 743-755.

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Western Blot Analysis of Stem Cell Markers

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The western blot experiment was performed as described previously 20 and the antibodies used were: IFITM1/2/3 (F12; sc‐374026; Santa Cruz Biotechnology), NANOG (sc‐293121; Santa Cruz Biotechnology), OCT3/4 (SC‐5279; Santa Cruz Biotechnology), SOX2 (AB5603, Millipore, Billerica, MA, USA), β‐actin (sc1616R; Santa Cruz Biotechnology) and IFITM3 (AF3377; R&D Systems). The protein bands were detected by Amersham ECL Prime western blot detection reagent (RPN2232; GE Healthcare, Chicago, IL, USA).

Fu Y., Zhou Z., Wang H., Gong P., Guo R., Wang J., Lu X., Qi F, & Liu L. (2017). IFITM1 suppresses expression of human endogenous retroviruses in human embryonic stem cells. FEBS Open Bio, 7(8), 1102-1110.

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Western Blot Analysis of Stem Cell Markers

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Cells were lysed in AM1 lysis buffer (Active Motif, Carlsbad, CA) and protein concentrations were measured with the BCA protein assay kit (Thermo Fisher Scientific Inc., Carlsbad, CA). Total protein (50 μg) was resolved by 125 g/L SDS-PAGE and transferred onto PVDF membranes. After being blocked in TBST (20 mmol/L Tris, 137 mmol/L NaCl, 1 g/L Tween20, pH 7.6) with 50 ml/L skim milk for 2 h at room temperature, membranes were incubated with CD44, CD133 (Miltenyi Biotech, San Diego, CA), Oct4 (Cell Signaling Technology, Danvers, MA), Nanog (Santa Cruz Biotechnology, Santa Cruz, CA), β-catenin (Cell Signaling Technology), and SOX2 (Cell Signaling Technology), and β-actin primary antibodies (diluted 1:500; Santa Cruz Biotechnology) for 2 h. Membranes were then washed three times with TBST solution, followed by incubation for 1 h with HRP-linked secondary antibodies (1:1000; Santa Cruz Biotechnology) at room temperature. Finally, membranes were visualized using the DAB reagent (Dako Corporation, Carpinteria, CA).

Zhang X., Hua R., Wang X., Huang M., Gan L., Wu Z., Zhang J., Wang H., Cheng Y., Li J, & Guo W. (2016). Identification of stem-like cells and clinical significance of candidate stem cell markers in gastric cancer. Oncotarget, 7(9), 9815-9831.

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Characterization of HNSCC Cancer Stem Cells

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HNSCC cell lines SNU1041 and FaDu were obtained from Korea Cell Line Bank (Seoul, Korea) and maintained in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Carlsbad, CA, USA), 10% FBS (Invitrogen), and penicillin/streptomycin (Invitrogen). Primary sphere cells (K3, K4, and K5) were isolated from surgical specimens from HNSCC patients, and the CSC properties were validated using a number of functional assays testing the self-renewal capability, stem cell marker expression, chemoresistance, and in vivo tumorigenicity, as reported previously (Lim et al, 2011 (link)). Primary sphere cells expanded in serum-free DMEM Ham's F-12 (DMEM/F12) medium supplemented with human recombinant basic fibroblast growth factor (bFGF; 10 ng/ml; R&D Systems, Minneapolis, MN, USA), N2 supplement (GIBCO, Franklin Lakes, NJ, USA), and epidermal growth factor (EGF; 10 ng ml⁻¹; R&D Systems). We purchased primary antibodies against proteins Oct4, Nanog, CD44, Snail, cyclin B1, vimentin, and ABCG2 from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Ki-67 from Chemicon International Inc. (Temecula, CA, USA), SOX2 from Abcam (Cambridge, UK), E-cadherin from Cell Signalling Technology (Beverly, MA, USA), and secondary antibodies, anti-rabbit IgG and anti-mouse IgG, from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Lee S.H., Oh S.Y., Do S.I., Lee H.J., Kang H.J., Rho Y.S., Bae W.J, & Lim Y.C. (2014). SOX2 regulates self-renewal and tumorigenicity of stem-like cells of head and neck squamous cell carcinoma. British Journal of Cancer, 111(11), 2122-2130.

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Western Blot Analysis of Pluripotency and Epithelial Markers

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Cells were washed twice in ice-cold PBS, and lysed in JS buffer (50 mM HEPES pH 7.5 containing 150 mM NaCl, 1% Glycerol, 1% Triton X100, 1.5 mM MgCl₂, 5 mM EGTA, 1 mM Na₃VO₄, and 1X protease inhibitor cocktail). Protein concentration was determined by the Bradford assay (BioRad, Milan, Italy) using bovine serum albumin as the standard, and equal amounts of proteins were analyzed by SDS-PAGE (12.5% acrylamide). Gels were electroblotted onto nitrocellulose membranes (G&E Healthcare, Milan, Italy). Membranes were blocked for 1 h with 5% non-fat dry milk in Tris Buffered Saline (TBS) containing 0.1% Tween-20, and incubated at 4°C overnight with the primary antibody. Detection was performed with peroxidase-conjugated secondary antibodies using an enhanced chemiluminescence system (ThermoEuroclone, Milan, Italy). Primary antibodies used were: anti-Zeb-1, -Oct 3/4, -Nanog, -cytokeratin 18, and -cytokeratin 8 (Santa Cruz Biotechnologies, MA, USA), anti-DNMT3b (Abcam, MA, USA), and anti-β-actin (Sigma Aldrich, Milan, Italy).

Roscigno G., Quintavalle C., Donnarumma E., Puoti I., Diaz-Lagares A., Iaboni M., Fiore D., Russo V., Todaro M., Romano G., Thomas R., Cortino G., Gaggianesi M., Esteller M., Croce C.M, & Condorelli G. (2015). MiR-221 promotes stemness of breast cancer cells by targeting DNMT3b. Oncotarget, 7(1), 580-592.

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Adenoviral Modulation of HGF Signaling

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MM cells were plated at a density of 5 × 10⁵ cells per well in 6-well tissue culture plates and grown in RPMI 1640/5% FBS. Cells were mock-infected or infected with Ad-LacZ or Ad-NK4 at a MOI of 100. After incubating for 30 h, the cells were treated with HGF (10 ng/ml) for 30 min and solubilized with lysis buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, plus protease and phosphatase inhibitors). The resulting cell lysates were separated by gel electrophoresis, transferred to PVDF membranes, and probed with primary antibodies followed by peroxidase-conjugated secondary antibodies. Protein bands were detected by means of an ECL Western analysis system (Amersham Biosciences, Inc.). An anti-β-gal antibody (Molecular Probes) or anti-HGFα (Santa Cruz) was used to detect adenovirus-mediated expression of LacZ and NK4, respectively. Protein bands were detected by using primary antibodies against Met, phospho-Met, AKT, phospho-AKT, ERK, phosphor-ERK, Nanog, Oct4, Myc (all from Santa Cruz Biotechnology), β-catenin (Cell Signaling Technology), and active β-catenin (anti-ABC, Millipore).

Deng X.B., Xiao L., Wu Y., Jin F., Mossman B., Testa J.R, & Xiao G.H. (2014). Inhibition of mesothelioma cancer stem-like cells with adenovirus-mediated NK4 gene therapy. International journal of cancer. Journal international du cancer, 137(2), 481-490.

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Ovarian Cancer Cell Characterization

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Fallopian tube epithelial cells (FTSEC or FTEC) were used as normal ovarian cells (generously provided by Dr. Amir Jazaeri) [13 ]. OV90 and SKOV3 cells were purchased from ATCC, isogenic A2780 (cisplatin-sensitive) and A2780/CP70 (cisplatin-resistant) cells were previously described [14 (link)]. OV90 and SKOV3 cells were cultured in 1:1 DMEM/F12 (Mediatech). FTSEC, A2780 and A2780/CP70 cells were cultured in RPMI [14 (link)]. All media were supplemented with 10% FBS and 1x Penicillin/Streptomycin. Carboplatin was from Sigma and Rad6 expression vector was obtained from Addgene [15 (link)]. The siRNAs used in this study were purchased from Dharmacon and the transfections were done using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. Antibodies specific to the following proteins were used: Gli1 (Cell Signaling Technology); GAPDH, ALDA1H1, BMI1, Nanog, OCT4, Myc, and β-Catenin (Santa Cruz Biotechnology); Rad6 (Bethyl Laboratories); H2B, H3K79me3 and SOX2 (Abcam); and Ub-H2B (Millipore).

Somasagara R.R., Tripathi K., Spencer S.M., Clark D.W., Barnett R., Bachaboina L., Scalici J., Rocconi R.P., Piazza G.A, & Palle K. (2015). Rad6 upregulation promotes stem cell-like characteristics and platinum resistance in ovarian cancer. Biochemical and biophysical research communications, 469(3), 449-455.

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Immunofluorescence Analysis of Pluripotency Markers

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Cells were fixed with 3.7 % formaldehyde solution for 1 h. at room temperature. After being washed twice with D-PBS, the cells were permeabilized with 0.1 % triton X-100 for 30 min and blocked in D-PBS supplemented with 1 % BSA solution, followed by overnight incubation at 4 °C with primary antibodies against Oct3/4 (Goat polyclonal, 1:200, Santa Cruz Biotechnology, Inc., CA, USA), Nanog (Goat polyclonal, 1:200, Santa Cruz Biotechnology), Sox2 (Rabbit polyclonal, 1:200, Santa Cruz), VASA (Rabbit polyclonal, 1: 100, Abcam, Cambridge, UK) and DAZL (Rabbit polyclonal, 1:100, Abcam) diluted in blocking solution. Slides were rinsed with D-PBS, and then incubated with FITC-conjugated donkey anti-goat IgG or goat anti-rabbit IgG (1:200, Jackson IR laboratories, Inc., PA, USA) for 45 min at 38.5 °C. Slides were then counterstaining with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature, mounted with Vectashield® (Vector Laboratories, Inc., CA, USA) and observed under a fluorescence microscope (Leica, Wetzlar, Germany).

Lee Y.M., Kim T.H., Lee J.H., Lee W.J., Jeon R.H., Jang S.J., Ock S.A., Lee S.L., Park B.W, & Rho G.J. (2016). Overexpression of Oct4 in porcine ovarian stem/stromal cells enhances differentiation of oocyte-like cells in vitro and ovarian follicular formation in vivo. Journal of Ovarian Research, 9, 24.

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