Colonies were fixed for 2 hours at room temperature with 4 % paraformaldehyde and then incubated at room temperature for 15 minutes with 1 % Triton X-100/phosphate-buffered saline (PBS). Cells were washed three times in PBS and blocked at 37 °C for over 3 hours with 4 % normal goat serum (Chemicon). Subsequently, cells were incubated at 4 °C overnight with primary antibody to Oct4 (1:500, Santa Cruz), SSEA4 (1:500, Life Technology), Nanog (1:500, Bethyl laboratories), TRA-1-60 (1:250, Life Technology), and TRA-1-81 (1:250, Life Technology). Cells were washed three times in PBS and incubated at 37 °C for 2 hours with goat anti-rabbit Alexa-Flour 594-conjugated (Life technologies) and goat anti-mouse Alexa-Fluor 488-conjugated (Life Technology) secondary antibodies (1:500 in 1 % normal goat serum in PBS). Unbound secondary antibodies were removed in three washes with PBS. Nuclei were identified by 1 μg/ml DAPI (Invitrogen) staining at room temperature for 5 minutes. Images were acquired using a confocal laser scanning microscope (LSM 510 META; Carl Zeiss).
Nanog
Manufactured by Fortis Life Sciences
Sourced in United States
Nanog is a protein that plays a key role in the maintenance and self-renewal of embryonic stem cells. It is a transcription factor that regulates the expression of genes involved in pluripotency and cell fate determination. Nanog is considered a critical marker for identifying and characterizing stem cells.
Automatically generated - may contain errors
Lab products found in correlation
Dppa3, by Abcam (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Stat3, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Ssea4, by Thermo Fisher Scientific (1 mentions)
Tra 1 60, by Thermo Fisher Scientific (1 mentions)
Tra 1 81, by Thermo Fisher Scientific (1 mentions)
Pstat3 y705, by Abcam (1 mentions)
Pierce ecl western blotting substrate, by Merck Group (1 mentions)
β catenin, by Santa Cruz Biotechnology (1 mentions)
P stat3, by Abcam (1 mentions)
Cw2333, by CWBIO (1 mentions)
Mcl 1, by Santa Cruz Biotechnology (1 mentions)
14 protocols using nanog
1
Pluripotent Stem Cell Characterization
2
Protein Analysis of ES Cells
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For protein analysis, ES cells were washed three times with PBS, and then were lysed with RIPA lysis buffer (Cowei) in the culture wells. Cell debris was removed by centrifugation. Protein concentration was determined by using the BCA Protein Assay kit (Thermo). Protein was collected and was separated by 10% SDS polyacrylamide gel electrophoresis and then transfer to PVDF membrane. The blots were probed with primary antibodies overnight at 4°C, and then for 1.5 hours at room temperature with appropriate secondary antibodies. Pierce ECL Western Blotting Substrate (Millipore) was used to detect the signal. The primary antibodies used include Stat3 (1:1000, Santa Cruz), p-Stat3-Y705 (1:1000, Abcam), Tbx3 (1:1000, Abcam), β-Actin (1:1000, Biotechnology), H3.1 (1:1000, Sungene), and Nanog (1:1000, Bethyl).
3
Western Blotting for Protein Expression
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To explore protein expression, western blotting was performed after the samples were harvested. Each group of 4T1 cells was lysed on ice in RIPA buffer (CW2333; cwbiotech) plus protease inhibitor cocktail and phosphatase inhibitors. Equivalent amounts of protein (BCA protein assay) from each sample were used for immunoblot analysis as described previously [21 (link)]. Proteins were examined with specific antibodies against Sox2 (Santa Cruz Biotechnology), Stat3 (Santa Cruz Biotechnology), p-Stat3 (Abcam), Oct4 (Santa Cruz Biotechnology), β-catenin (Santa Cruz Biotechnology), Nanog (Bethyl), and β-actin (Santa Cruz Biotechnology).
4
Western Blot Analysis of Cellular Proteins
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Lysate extracted from a total of 1 × 105 cells was used to perform Western blots analysis. Primary antibodies against pEGFR (1:3000; Cell Signaling Technology, Danvers, MA, USA, 3777 S), EGFR (1:3000; Cell Signaling Technology, 4267 S), MCL1 (1:3000; Santa Cruz Biotechnology, Paso Robles, CA, USA, sc-819), ATG7 (1:3000; Cell Signaling Technology, 8558 S), LC3B (1:3000; Cell Signaling Technology, 2775 S), EGF (1:1000; Abcam, Cambridge, UK, ab206106), NANOG (1:3000; Bethyl Laboratories, Montgomery, TX, USA, A300-379A), TRPV1 (1:3000; Abcam, ab6166), FLAG (1:5000; Medical & Biological Laboratories, Nagoya, JPN, M185-3L), pAKT (1:3000; Cell Signaling Technology, 9271), AKT1 (1:3000; Cell Signaling Technology, 9272), and β-actin (1:5000; Medical & Biological Laboratories, M177-3) were used. Western blotting was followed by incubation with the appropriate secondary antibodies conjugated to horseradish peroxidase (HRP), anti-rabbit IgG-HRP (1:5000; Enzo, Farmingdale, NY, USA, ADI-SAB-300-J), and anti-mouse IgG-HRP (1:5000; Enzo, ADI-SAB-100-J). The immunoreactive bands were developed with the chemiluminescence ECL Detection System (GE Healthcare, Chicago, IL, USA), and signals were detected using a luminescent image analyzer (LAS-4000 Mini, Fujifilm, JPN).
5
Immunofluorescence analysis of mESC differentiation
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After fixation for 20 min with 4% paraformaldehyde at room temperature, mESCs were permeabilized with 0.0125% Triton X-100. Then, the mESCs were incubated with 1% BSA and incubated overnight with a primary antibody specific to Tuj1 (1:500; Sigma, St. Louis, MO, USA), Map2 (1:500; Cell Signaling Technology, USA), Brachyury (1:500; Santa Cruz, CA, USA), Foxa2 (1:500; Santa Cruz, CA, USA), Oct4 (1:500; Santa Cruz, CA, USA), or Nanog (1:500; Bethyl Lab, Montgomery, TX, USA). After washing with PBS, mESCs were incubated at room temperature for 90 min with specific secondary antibodies (1:1000; Invitrogen, Carlsbad, CA, USA). Counterstaining was performed with DAPI (5 mg/ml, Sigma, St. Louis, MO, USA). Cells ere imaged with a Nikon eclipse Ti. The three-color images were saved as a tif file format and merged with Adobe Photoshop software.
6
Immunofluorescence Profiling of Stem Cells
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Colonies were dissociated into single cells and resuspended in corresponding culture medium at a density of approximately 5 × 106 cells/ml. Cells were fixed and permeabilized by 3.7% paraformaldehyde and 0.1% Triton X-100 in PBS, blocked by 4% BSA, and then incubated with primary antibodies including Dppa3 (1:500; Abcam), Nanog (1:500; Bethyl), or isotype-matched negative control. Alexa Fluor 594 goat anti-mouse IgG (6 μg/ml; Invitrogen) was used as secondary antibodies. Cells were then analyzed using FACSAria Cell Sorter (FACStar Plus Flow Cytometer; Becton-Dickson).
7
Protein Expression Analysis by Western Blot
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Total protein was extracted by using RIPA lysis buffer (ThermoFisher Scientific). Cell lysates were separated by SDS-PAGE and transferred onto a PVDF (Millipore). PVDF was probed with primary antibodies against Nanog (Bethyl), Bcl3, Sox2, Oct4, p53, GAPDH, β-actin (Santacruz) followed by the application of HRP-conjugated secondary antibodies. HRP was detection by the application of ECL reagent (Millipore) to PVDF and exposure to x-ray films.
8
Whole Cell Extraction for Western Blotting
9
Protein Expression Analysis Protocol
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Cells were scraped/trypsinized, washed in PBS and incubated for 20 min in cold RIPA buffer without SDS. Protein concentrations were determined using Bradford Dye (Bio-Rad). Total proteins (∼10 ug) were separated on SDS–PAGE gels and transferred to PVDF membranes (Millipore). Membranes were probed with specific primary antibodies followed by HRP-conjugated secondary antibodies and developed with ECL (Amersham). Primary antibodies were: Oct4 (sc9081, Santa Cruz), Nanog (A300-398A, Bethyl), Actin (sc1615, Santa Cruz), Sox2 (sc17320, Santa Cruz) and Esrrb (300–748, Novus Biologicals). Amido-black was used to detect core histones. Quantification of protein bands was performed using Adobe Photoshop. Relative protein level differences were calculated by normalization to Actin levels and shown relative to empty-vector transfected sample.
10
mESC Culture and Antibody Validation
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We used the mESC line E14Tg2a in this study. Cells were cultured on 0.1% gelatin-coated (Millipore) plates in DMEM (Life Technologies) supplemented with 15% fetal bovine serum (Sigma), penicillin/streptomycin, nonessential amino acid, sodium pyruvate, GlutaMax, β-mercaptoethanol (Life Technologies), and 1000 U/mL LIF (ESGRO, Millipore) unless specified. Antibodies used in this study were 5hmC (Active Motif, 39769), Oct4 (Santa Cruz Biotechnology, sc-8628), Nanog (Bethyl Laboratories, IHC-00205), Sox2 (Millipore, Ab5603), Tet1 (Ito et al. 2010 (link)), Tet2 (described below), Tet3 (Gu et al. 2011 (link)), Zscan4 (Millipore, Ab4340), Suz12 (Cao et al. 2002 (link)), Ring1b (Cell Signaling, 5694), Ezh2 (Cell Signaling, 5246), and Kap1 (Cell Signaling, 4123). Tet2 antibody was generated using HIS-tagged recombinant Tet2 2-374 expressed in Escherichia coli and purified using Talon superflow metal affinity resin (Clontech). Rabbit immunization was carried out by Pocono Rabbit Farm and Laboratory, Inc. The antiserum was affinity-purified using immunization antigen. The specificity of the affinity-purified antibody was confirmed using extracts from the control and Tet2 knockdown mESCs.
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