NANOG Antibody

What is the functional role of NANOG in stem cell pluripotency?

NANOG functions as a transcription regulator critically involved in inner cell mass and embryonic stem (ES) cell proliferation and self-renewal. It imposes pluripotency on ES cells and prevents their differentiation towards extraembryonic endoderm and trophectoderm lineages . NANOG also blocks bone morphogenetic protein-induced mesoderm differentiation by physically interacting with SMAD1 and interfering with the recruitment of coactivators to active SMAD transcriptional complexes .

Research has demonstrated that NANOG can act as both a transcriptional activator and repressor, binding optimally to specific DNA consensus sequences: 5'-TAAT[GT][GT]-3' or 5'-[CG][GA][CG]C[GC]ATTAN[GC]-3' . NANOG can also bind to the POU5F1/OCT4 promoter and autoregulatе its expression in differentiating ES cells through interaction with ZNF281/ZFP281, leading to recruitment of the NuRD complex and subsequent repression .

Importantly, NANOG-NANOG homodimerization has been shown to be critical for stem cell pluripotency , highlighting the complex molecular mechanisms through which NANOG maintains the undifferentiated state.

Key considerations for species-specific antibody selection:

• Human NANOG maps to gene locus 12p13.31, while mouse NANOG maps to gene loci 6 F2

• Mouse embryonic NANOG expression is detected specifically in the inner cell mass of the blastocyst

• Several antibodies are specifically validated for human samples (AF1997) , mouse samples (AF2729) , or both (variable cross-reactivity)

• When working with non-human, non-mouse models, homology prediction should be considered, but validation is essential

Researchers should verify that their chosen antibody has been validated for their species of interest and consider the specific epitope recognized by the antibody in relation to protein conservation across species .

How can I optimize NANOG ChIP experiments for studying pluripotency gene networks?

For optimal NANOG chromatin immunoprecipitation (ChIP) results, consider the following protocol details from the search information:

• Sample preparation: Fix embryonic stem cells using formaldehyde, resuspend in lysis buffer, and sonicate to shear chromatin to appropriate fragment sizes .

• Antibody amounts: Use approximately 5 μg of NANOG antibody per 5 x 10^6 cells for immunoprecipitation . For validated antibodies like Cell Signaling Technology's Nanog Antibody (#3580), use 20 μl of antibody and 10 μg of chromatin (approximately 4 x 10^6 cells) per IP .

• Immunoprecipitation technique: After adding the NANOG antibody (such as AF1997), incubate for 15 minutes in an ultrasonic bath, followed by addition of a secondary antibody (e.g., Biotinylated Anti-Goat IgG) .

• Complex capture: Capture immunocomplexes using streptavidin ferrofluid (e.g., 50 μL of MagCellect Streptavidin Ferrofluid) and purify DNA using chelating resin solution .

• Target detection: For detection of NANOG-regulated genes, standard PCR or qPCR can be used with specific primers like Human Nanog Primer Pair (such as RDP-320-025) .

• Controls: Always include a negative control antibody (e.g., AB-108-C) in parallel reactions to assess background and specificity .

This approach has been successfully used to detect the NANOG promoter and study NANOG-regulated genes in human embryonic stem cells .

What are the critical considerations when analyzing NANOG splice variants and protein isoforms?

Alternative splicing of NANOG produces multiple protein variants with distinct functional properties in pluripotency maintenance and self-renewal capacity . When studying these variants:

• Variant-specific detection: Design primers that specifically amplify different NANOG transcript variants. For quantification, qRT-PCR can be performed to determine relative expression levels of different transcripts across cell lines .

• Expression quantification: Normalize expression to housekeeping genes like Ywhaz when comparing variant expression across different cell lines (e.g., J1, V6.5, RF8, E14Tg2a) .

• Functional assessment: To evaluate the functional differences between NANOG variants, conduct alkaline phosphatase (AP) staining assays with and without LIF (Leukemia Inhibitory Factor) for 8 days .

• Colony morphology analysis: Score colonies for both AP activity and morphology to distinguish between pluripotent and differentiated states when comparing the effects of different NANOG variants .

• Antibody epitope consideration: Verify that your chosen antibody can detect your variant of interest by checking if the epitope falls within the variant-specific region. Some antibodies are raised against specific domains that may be absent in certain splice variants .

Research has shown that alternative splicing results in NANOG protein variants with attenuated capacities for self-renewal and pluripotency in ES cells , making proper variant identification critical for accurate interpretation of results.

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

The search results don't directly address post-translational modifications (PTMs) of NANOG, but based on general principles and the information provided:

• Western blot considerations: NANOG detection by Western blot typically reveals a band at approximately 40-45 kDa , but PTMs like phosphorylation, SUMOylation, or ubiquitination may alter migration patterns. When unexpected band sizes appear, consider whether PTMs might be responsible.

• Reduction conditions: Western blot experiments with NANOG are typically conducted under reducing conditions , which may affect the detection of certain PTMs or protein conformations.

• Buffer selection: When performing Western blot for NANOG, buffer selection can be important. For example, AF1997 antibody has been validated using Immunoblot Buffer Group 9 , which may optimize detection of certain protein states.

• Cellular context: NANOG function changes during differentiation, likely involving PTMs. When studying NANOG during differentiation processes, consider using antibodies that recognize the protein regardless of common modification states.

• Dimerization detection: Since NANOG-NANOG homodimerization is critical for stem cell pluripotency , using non-reducing conditions might help preserve and detect these functionally important dimeric forms.

What are the best fixation and permeabilization methods for NANOG immunostaining in different sample types?

Based on the search results, several validated protocols exist for NANOG immunostaining:

For immunocytochemistry of cultured cells:

• Use immersion fixation for embryonic stem cells and iPSCs

• For flow cytometry, fix cells with Flow Cytometry Fixation Buffer (e.g., FC004) and permeabilize with Flow Cytometry Permeabilization/Wash Buffer I (e.g., FC005)

• For staining intensity optimization, apply antibody at 10 μg/mL for 3 hours at room temperature

• Counterstain with DAPI for nuclear visualization, as NANOG shows nuclear localization

For paraffin-embedded tissue sections:

• Perform heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (e.g., CTS013) before primary antibody incubation

• Apply NANOG antibody at 1 μg/mL for 1 hour at room temperature

• For visualization, use an appropriate HRP polymer system (e.g., Anti-Goat IgG VisUCyte™ HRP Polymer Antibody)

• Develop with DAB (brown) and counterstain with hematoxylin (blue)

For embryoid bodies:
Special care should be taken when staining embryoid bodies derived from ES cells, as NANOG expression will vary throughout the structure. Successful detection has been achieved using 10 μg/mL antibody with NorthernLights™ 557-conjugated secondary antibodies .

How can I improve specificity and reduce background in NANOG Western blots?

For optimal NANOG Western blot results:

• Sample preparation: Obtain lysates from appropriate positive control cells such as BG01V human embryonic stem cells, Tera-2 human embryonic lung carcinoma cell line , or F9 embryonal carcinoma cells for mouse studies .

• Negative controls: Include lysates from differentiated cells or cell lines known not to express NANOG, such as NIH3T3 cell line or mouse spleen for mouse studies .

• Membrane selection: Use PVDF membrane, which has been successfully used in validated protocols .

• Antibody dilution: For optimal signal-to-noise ratio, use antibody at recommended dilutions (typically 1 μg/mL for AF1997 or 1:1000 for Cell Signaling Technology antibody #3580 ).

• Expected band size: Look for a specific band at approximately 40 kDa for human NANOG or ~45 kDa for mouse NANOG in F9 lysate .

• Buffer optimization: Use recommended buffer systems, such as Immunoblot Buffer Group 9 for AF1997 antibody .

• Secondary antibody selection: Choose appropriate secondary antibodies that minimize cross-reactivity, such as HRP-conjugated Anti-Goat IgG for goat primary antibodies .

These approaches have been validated in published protocols and should help improve specificity while reducing background interference.

What strategies can be used to validate NANOG antibody specificity for research applications?

Thorough validation of NANOG antibodies is critical due to the protein's importance in stem cell research. Based on the search results, consider these validation strategies:

• Multi-application concordance: Verify consistent results across different applications (WB, ICC/IF, ChIP, flow cytometry) with the same antibody .

• Multiple antibody approach: Use antibodies from different sources or raised against different epitopes of NANOG to confirm findings .

• Positive controls: Include well-characterized NANOG-expressing cells such as:

  • BG01V human embryonic stem cells

  • Tera-2 human embryonic lung carcinoma cells

  • F9 embryonal carcinoma cells (for mouse studies)

  • D3 mouse embryonic stem cell line

• Negative controls: Include known NANOG-negative samples:

  • Differentiated cells

  • NIH3T3 cell line (for mouse studies)

  • Mouse spleen tissue

• Recombinant protein controls: Compare against recombinant NANOG proteins of defined regions:

  • Human NANOG Trp153-Val305 (Accession # ABZ92376)

  • Mouse NANOG Trp154-Leu262 (Accession # Q80Z64)

• Genetic approaches: Use NANOG knockout or knockdown cells as negative controls, or overexpression systems as positive controls .

• Peptide competition: Pre-incubate antibody with immunizing peptide to demonstrate binding specificity.

• Cross-species reactivity testing: Verify whether antibodies claiming cross-reactivity actually detect the protein across species with expected size and localization patterns .

How can NANOG antibodies be utilized to study reprogramming mechanisms in iPSC generation?

NANOG antibodies are valuable tools for monitoring the reprogramming process during induced pluripotent stem cell (iPSC) generation:

• Reprogramming factor detection: NANOG is one of the factors that contribute to reprogramming differentiated cells to an induced pluripotent stem cell state . Antibodies can monitor its expression during the reprogramming process.

• Alternative reprogramming assessment: Research has demonstrated that iPS cells can be generated using expression plasmids expressing NANOG, Sox2, KlfF4, and c-Myc, eliminating the need for virus introduction . NANOG antibodies can verify successful expression from these plasmids.

• Pluripotency verification: NANOG antibodies are used to confirm pluripotency in newly generated iPSC lines, as demonstrated in multiple studies:

  • NANOG was detected in immersion fixed ADLF1 and FAB2 induced pluripotent stem cell lines using antibodies at 10 μg/mL

  • PluriTest analysis combined with NANOG antibody-based flow cytometry has been used to verify pluripotency in iPS-OX1-18, iPS-OX1-19, and iPS-OX1-23 cell lines

• Co-expression analysis: NANOG antibodies can be combined with antibodies against other pluripotency markers (Oct4, SOX2, SSEA4, TRA-1-60) to comprehensively characterize the pluripotent state of reprogrammed cells .

• Partial reprogramming detection: Different expression levels of NANOG can indicate partial versus complete reprogramming, with antibody-based quantitative methods like flow cytometry providing valuable insights into reprogramming efficiency .

What are the best approaches for studying NANOG protein interactions using co-immunoprecipitation?

For effective NANOG co-immunoprecipitation experiments:

• Antibody selection for IP: Several antibodies have been validated for immunoprecipitation of NANOG, including mouse monoclonal [NNG-811] antibody (ab62734) and other commercial antibodies specified for IP applications.

• Buffer considerations: Standardized lysis buffers that maintain protein interactions while effectively solubilizing nuclear proteins should be used, as NANOG is primarily localized to the nucleus .

• Known interaction partners: Design experiments to detect established NANOG interaction partners such as:

  • SMAD1 (NANOG blocks BMP-induced differentiation by physically interacting with SMAD1)

  • ZNF281/ZFP281 (NANOG interacts with this factor when binding to its own promoter)

  • Other pluripotency transcription factors like OCT4 and SOX2

• Controls for specificity: Always include appropriate negative controls:

  • IgG control antibodies of the same species and isotype

  • Lysates from cells not expressing NANOG

  • When possible, NANOG-knockout or knockdown cells

• Detection methods: Western blot analysis using antibodies against suspected interaction partners following NANOG immunoprecipitation can confirm interactions.

• Directionality testing: Perform reciprocal co-IP experiments, precipitating with antibodies against the suspected interaction partner and blotting for NANOG.

• Truncation mutants: To map interaction domains, consider using the truncated NANOG mutants described in the research literature , which can help identify specific regions required for protein-protein interactions.

How can NANOG antibodies be employed to study the dynamics of pluripotency loss during differentiation?

NANOG antibodies provide powerful tools for tracking pluripotency dynamics during differentiation:

• Time-course analysis: Monitor NANOG expression over time during differentiation protocols. Research has used time-lapse imaging of differentiating ES cells treated with retinoic acid for up to 40 hours to track changes in nuclear proteins during differentiation .

• Co-staining approaches: Combine NANOG antibodies with differentiation markers to identify transitional cell states. For example, staining for NANOG alongside early lineage markers can reveal the heterogeneity of differentiation responses .

• Quantitative assessment: Use flow cytometry with NANOG antibodies to quantify the percentage of cells maintaining pluripotency during differentiation protocols. This approach allows for precise measurement of population dynamics .

• Chromatin dynamics: ChIP experiments using NANOG antibodies at different time points during differentiation can reveal how NANOG occupancy at target genes changes over time, providing insights into the molecular mechanisms of pluripotency loss .

• Single-cell variation: Immunofluorescence with NANOG antibodies can reveal cell-to-cell variability in pluripotency factor expression during differentiation, which has been shown to influence differentiation potential .

• Embryoid body analysis: NANOG antibodies have been successfully used to analyze differentiation within embryoid bodies, revealing spatial patterns of pluripotency factor loss that may mimic aspects of embryonic development .

These approaches can provide valuable insights into the mechanisms controlling the exit from pluripotency and entry into differentiation pathways.