NANOG Antibody

What is NANOG protein and why is it significant in research?

NANOG is a critical homeodomain-containing transcription factor that plays an essential role in maintaining pluripotency and self-renewal of embryonic stem cells, thereby preventing differentiation. Structurally, NANOG comprises a conserved homeodomain that facilitates specific DNA binding, as well as regions that enable interactions with other key proteins involved in the pluripotency network, such as Oct4 and Sox2. These interactions are vital for forming transcriptional complexes that regulate the expression of genes necessary for sustaining the undifferentiated state of stem cells . NANOG's significance lies in its pivotal role in orchestrating gene expression programs that maintain cellular pluripotency and drive self-renewal, making it a key research target for understanding stem cell biology and developmental processes.

What types of NANOG antibodies are available for research applications?

Research laboratories can utilize several types of NANOG antibodies, each with specific applications and advantages:

• Monoclonal antibodies (e.g., H-2): Mouse monoclonal IgG2a kappa light chain antibodies engineered to detect NANOG from mouse, rat, and human origins. These antibodies target specific epitopes, such as the C-terminus (amino acids 151-305) of human NANOG .

• Polyclonal antibodies (e.g., AF1997): Goat anti-human NANOG antigen affinity-purified polyclonal antibodies that recognize specific regions such as recombinant human NANOG Trp153-Val305 .

The choice between monoclonal and polyclonal antibodies depends on the research application, with monoclonals offering high specificity for a single epitope and polyclonals providing broader detection capabilities across multiple epitopes of the NANOG protein.

What are the key applications of NANOG antibodies in stem cell research?

NANOG antibodies serve multiple crucial roles in stem cell research:

• Characterization of pluripotent stem cells: NANOG antibodies can verify stemness in embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and embryonal carcinoma cells through detection of this key pluripotency marker .

• Monitoring differentiation dynamics: Researchers use NANOG antibodies to track changes in pluripotency during directed differentiation protocols, as NANOG expression decreases during lineage commitment .

• Chromatin immunoprecipitation (ChIP) assays: These assays identify NANOG binding sites in the genome, illuminating its role in transcriptional regulation networks. Studies have successfully used NANOG antibodies to immunoprecipitate NANOG/DNA complexes in human embryonic stem cells .

• Cancer research: NANOG expression has been detected in certain cancers like testicular cancer, suggesting roles in oncogenesis and cancer stem cell maintenance .

• Developmental biology: NANOG antibodies help trace embryonic development patterns, with studies showing specific staining in developing central nervous system tissues of mouse embryos .

What are the optimal methodologies for using NANOG antibodies in Western blot applications?

For optimal Western blot results with NANOG antibodies, researchers should follow these methodological considerations:

• Sample preparation: For stem cell lysates, use complete lysis buffers containing protease inhibitors to prevent degradation of NANOG protein. RIPA or NP-40 based buffers are commonly effective.

• Loading controls: Always include appropriate loading controls (e.g., GAPDH, β-actin) to normalize NANOG expression levels .

• Expected molecular weight: Prepare to visualize NANOG at approximately 40-55 kDa, depending on the cell type and antibody used. Research data shows NANOG detected at approximately 40 kDa in BG01V human embryonic stem cells and Tera-2 human embryonic lung carcinoma cell lines using the AF1997 antibody . In iPSC lysates, NANOG has been detected at approximately 55 kDa using the same antibody in Simple Western™ systems .

• Reducing conditions: NANOG Western blots generally perform optimally under reducing conditions. The R&D Systems data specifically notes that experiments were conducted "under reducing conditions and using Immunoblot Buffer Group 9" .

• Secondary antibody selection: Choose appropriate conjugated secondary antibodies that match the host species of your primary antibody (e.g., HRP-conjugated Anti-Goat IgG for goat primary antibodies) .

How can researchers optimize immunofluorescence protocols for NANOG detection in pluripotent stem cells?

For optimal immunofluorescence detection of NANOG in pluripotent stem cells, researchers should implement these methodological approaches:

• Fixation: Use 4% paraformaldehyde for adequate fixation while preserving epitope accessibility. The protocol should typically involve immersion fixation as demonstrated in multiple successful experiments with BG01V human embryonic stem cells and induced pluripotent stem cell lines .

• Nuclear localization: Optimize for nuclear staining since NANOG primarily localizes to the nucleus in undifferentiated stem cells. Always include nuclear counterstaining (e.g., DAPI) to confirm nuclear localization .

• Antibody concentration: Titrate antibody concentrations; successful protocols have used concentrations around 10 μg/mL for primary NANOG antibodies in stem cell immunofluorescence .

• Incubation conditions: Perform primary antibody incubation for approximately 3 hours at room temperature as demonstrated in protocols using Goat Anti-Human NANOG antibodies on various stem cell types .

• Appropriate controls: Include negative controls (secondary antibody only) and positive controls (established pluripotent stem cell lines with known NANOG expression) .

• Co-staining approaches: Consider simultaneous staining for other pluripotency markers (Oct4, Sox2, SSEA4) to validate stemness comprehensively, as described in protocols where H7 cells expressed multiple markers including NANOG, Oct4, SSEA4, and TRA-1-60 .

What are the best practices for using NANOG antibodies in chromatin immunoprecipitation (ChIP) assays?

When performing ChIP assays with NANOG antibodies, researchers should follow these best practices for optimal results:

• Cross-linking optimization: Use formaldehyde (typically 1%) to cross-link protein-DNA complexes in living cells. The search results show successful ChIP protocols using formaldehyde fixation of BG01V human embryonic stem cells .

• Sonication parameters: Optimize sonication conditions to generate DNA fragments of 200-500 bp. The example protocol mentions sonication to shear chromatin prior to immunoprecipitation .

• Antibody selection and concentration: Choose ChIP-validated antibodies at appropriate concentrations. The documented protocol used 5 μg of Goat Anti-Human NANOG Antigen Affinity-purified Polyclonal Antibody for immunoprecipitation of NANOG/DNA complexes .

• Immunoprecipitation conditions: The search results describe a protocol using 15-minute incubation in an ultrasonic bath for immunoprecipitation, followed by Biotinylated Anti-Goat IgG Secondary Antibody .

• Magnetic bead capture: The example protocol employed MagCellect Streptavidin Ferrofluid (50 μL) to capture immunocomplexes, which is an efficient method for isolating antibody-bound chromatin .

• DNA purification and analysis: After capture, DNA was purified using chelating resin solution, and the NANOG promoter was detected by standard PCR to verify the success of the ChIP procedure .

What are common issues with NANOG antibody specificity and how can researchers address them?

Researchers frequently encounter specificity issues with NANOG antibodies that can be addressed through systematic approaches:

• Cross-reactivity concerns: NANOG antibodies may cross-react with NANOG pseudogenes or related proteins. To address this:

  • Validate antibodies using positive controls (embryonic stem cells) and negative controls (fully differentiated cells)

  • Employ siRNA knockdown experiments to confirm specificity, as demonstrated in studies where RNAi against NANOG produced specific downregulation of the target protein as confirmed by Western blot

• Isoform detection variability: Different antibodies may preferentially detect specific NANOG isoforms. To manage this:

  • Select antibodies raised against conserved regions if detecting all NANOG variants is desired

  • For monoclonal antibodies like H-2, note that they are raised against specific regions (e.g., amino acids 151-305 at the C-terminus of human NANOG)

• Species-specific considerations: Ensure the antibody recognizes NANOG from your species of interest. The search results indicate that some antibodies, like the H-2 monoclonal antibody, are engineered to detect NANOG from multiple species including mouse, rat, and human origins .

How can researchers optimize NANOG antibody performance in different cell types and tissues?

Optimizing NANOG antibody performance across different biological samples requires methodological adaptations:

• Embryonic stem cells vs. somatic tissues: NANOG expression varies dramatically between pluripotent and differentiated cells. Studies demonstrate successful NANOG detection in:

  • BG01V human embryonic stem cells (nuclear localization)

  • Human testicular cancer tissues (nuclear localization)

  • Developing mouse embryo CNS tissues (nuclear localization)

  • Normal human testis (cytoplasmic localization in developing sperm cells)

• Tissue-specific protocol modifications:

  • For paraffin-embedded tissues: Implement heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic prior to antibody incubation, as used successfully in human testicular cancer and normal testis tissues

  • For frozen sections: The example from 13 d.p.c. mouse embryos also employed heat-induced epitope retrieval before primary antibody incubation

• Cell-line specific modifications:

  • For cancer cell lines: Research has shown that DHT treatment can increase NANOG expression in prostate cancer cell lines (LNCaP and PC-3), requiring protocol adjustments to account for treatment-induced expression changes

How can NANOG antibodies be utilized in studying cellular reprogramming and iPSC generation?

NANOG antibodies serve crucial functions in cellular reprogramming research:

• Reprogramming verification: NANOG antibodies confirm successful generation of induced pluripotent stem cells (iPSCs). Flow cytometry data shows expression of pluripotency markers including NANOG in validated iPSC lines, establishing this as a definitive verification approach .

• Temporal expression analysis: During reprogramming, researchers can track NANOG expression dynamics using techniques like qRT-PCR complemented by immunoblotting. This approach was demonstrated in experiments where cells were collected on days 2, 4, 6, 8, and 10 after transfection with reprogramming factors to monitor NANOG expression over time .

• Self-replicating RNA reprogramming: In advanced reprogramming approaches, NANOG antibodies help verify protein expression from self-replicating RNA constructs. Research data shows immunoblot analysis of NANOG in cells transfected with 5F-srRNA on days 6 and 10, with iPSC clones serving as positive controls .

• Quality control of iPSC lines: Immunocytochemistry with NANOG antibodies helps assess the quality and pluripotency status of established iPSC lines across multiple passages, as demonstrated in studies of undifferentiated H7 hESC colonies .

What insights can NANOG antibodies provide about cancer stem cells and tumor development?

NANOG antibodies have revealed important connections between pluripotency networks and cancer biology:

• Cancer stem cell identification: Immunohistochemistry using NANOG antibodies has successfully identified cells with stem-like properties in human testicular cancer, where specific staining was localized to cell nuclei. This suggests NANOG as a potential cancer stem cell marker in certain malignancies .

• Hormone effects on stemness factors: Studies using immunoblotting and immunocytochemistry demonstrate that dihydrotestosterone (DHT) increases NANOG expression in prostate cancer cell lines (LNCaP and PC-3), suggesting hormonal regulation of stem cell factors in prostate cancer .

• Differential expression patterns: NANOG expression in normal tissues versus tumors provides insights into malignant transformation processes. For example, in normal human testis, NANOG was localized to the cytoplasm in developing sperm cells, while in testicular cancer, NANOG showed nuclear localization .

• Developmental cancer connections: NANOG antibody staining in both embryonic tissues (e.g., developing CNS) and certain cancers highlights potential developmental pathway reactivation in malignancies .

How can NANOG antibodies be used in multiplex immunophenotyping of stem cell populations?

Advanced multiplex immunophenotyping using NANOG antibodies enables comprehensive characterization of stem cell heterogeneity:

• Co-expression analysis with core pluripotency factors: NANOG antibodies can be combined with antibodies against other pluripotency markers (Oct4, Sox2, SSEA4, TRA-1-60) to comprehensively assess stemness. Research data shows successful co-expression analysis in H7 cells passaged on different feeder cell types .

• Flow cytometry applications: NANOG antibodies enable quantitative assessment of pluripotency marker expression in cell populations. Flow cytometric analysis has been successfully used to evaluate NANOG expression alongside OCT4, SOX2, and SSEA4 in undifferentiated stem cell lines .

• Single-cell heterogeneity analysis: Advanced protocols combine NANOG antibodies with other markers to assess cell-to-cell variation within stem cell populations. This approach was demonstrated in studies examining differentiation characteristics of individual stem cells derived from the NTERA2.Tom cell line .

• Tracking pluripotency during differentiation: Multiplex immunostaining with NANOG antibodies helps monitor the gradual loss of pluripotency markers during differentiation protocols, providing insights into lineage commitment dynamics .

How should researchers interpret varying NANOG expression levels across different experimental contexts?

Interpreting NANOG expression requires considering multiple contextual factors:

• Cellular heterogeneity in pluripotent cultures: Pluripotent stem cell populations often display heterogeneous NANOG expression. Studies using flow cytometry have characterized this variation, showing that even verified pluripotent cell lines can contain subpopulations with different NANOG expression levels .

• Differentiation stage effects: Interpret NANOG expression in relation to differentiation status. During cellular reprogramming with self-replicating RNA, NANOG expression increases progressively over time (days 2-10), reflecting the gradual acquisition of pluripotency .

• Microenvironment influences: Culture conditions significantly impact NANOG expression. Research comparing H7 hESCs cultured on human foreskin fibroblast (hFF) feeder cells versus mouse embryonic fibroblast (MEF) feeder cells demonstrated maintenance of NANOG expression despite different culture environments .

• Relationship to other pluripotency factors: Always interpret NANOG expression in relation to other stemness markers. For instance, RNAi experiments revealed that NANOG knockdown cells maintained OCT4 positivity, demonstrating the complexity of pluripotency network regulation .

What validation techniques should accompany NANOG antibody-based experiments?

Robust validation approaches are essential for NANOG antibody experiments:

• Multiple detection methodologies: Validate findings using complementary techniques. Research protocols frequently combine:

  • Protein detection (Western blot, immunocytochemistry)

  • Transcript analysis (qRT-PCR)

  • Functional assays (ChIP)

• Knockdown/knockout controls: RNA interference experiments targeting NANOG provide essential validation controls. Studies have demonstrated NANOG knockdown efficiencies between 60% and >90%, with corresponding decreases in protein levels as confirmed by Western blot .

• Multiple antibody validation: Use different antibodies (monoclonal and polyclonal) targeting distinct NANOG epitopes to confirm specificity. The search results describe both mouse monoclonal antibodies targeting amino acids 151-305 and goat polyclonal antibodies recognizing Trp153-Val305 .

• Pluripotency correlation: Validate NANOG detection in relation to established pluripotency metrics. PluriTest analysis of transcriptome array data can confirm that cells expressing NANOG cluster with verified pluripotent stem cells rather than differentiated cells .

• Species-appropriate positive controls: Human seminoma tissue sections serve as excellent positive controls for NANOG antibodies, as demonstrated in immunocytochemical validation studies .

How do post-translational modifications affect NANOG detection with antibodies?

Post-translational modifications significantly impact NANOG antibody detection:

• Molecular weight variations: NANOG appears at different molecular weights across detection methods and cell types. While the theoretical molecular weight is around 34-35 kDa, actual detection often occurs at:

  • Approximately 40 kDa in Western blots of embryonic stem cells and carcinoma cell lines

  • Approximately 55 kDa in Simple Western analysis of iPSC lysates
    These variations likely reflect post-translational modifications and should be considered when interpreting results.

• Phosphorylation effects: NANOG undergoes phosphorylation that can affect antibody binding. Research indicates that phosphorylation status can influence NANOG protein stability and function, potentially affecting detection efficiency.

• Sumoylation considerations: NANOG can undergo sumoylation, which may alter its molecular weight and detection profile in certain experimental contexts. This modification should be considered when unexpected banding patterns appear in Western blot analyses.

• Sample preparation impact: Preserving post-translational modifications requires appropriate lysis buffers and handling protocols. The use of phosphatase inhibitors in sample preparation may be crucial for consistent detection of modified NANOG protein.