NR2F1 Antibody, HRP conjugated

What is NR2F1 and what are its key biological functions?

NR2F1 is an orphan nuclear receptor belonging to the steroid/thyroid hormone receptors superfamily. It contains a DNA-binding domain with two conserved zinc-finger motifs and a ligand-binding domain (LBD) . NR2F1 functions as a transcriptional regulator through multiple mechanisms: it can bind directly as a dimer to direct repeats on DNA and recruit coactivator or corepressor complexes, or it can act indirectly as a cofactor to other nuclear receptors . Depending on cellular context, NR2F1 can both activate and repress transcription of target genes . Beyond direct transcriptional regulation, NR2F1 plays a significant epigenetic role by mediating global histone modifications through interactions with chromatin-remodeling enzymes .

How does NR2F1 expression vary across tissue types and disease states?

NR2F1 shows notable expression differences between normal and malignant tissues. In head and neck squamous cell carcinoma (HNSCC) patients, NR2F1 expression is typically absent or low in primary tumors, recurrent tumors, and metastases compared to benign adjacent oral mucosa . Studies have shown that approximately 85% of HNSCC primary tumors (n=41) exhibit downregulation of NR2F1 . Quantitative analysis has revealed that while normal adjacent tissues show high NR2F1 expression (7-66% range), only 1.5±2.5% of tumor cells in primary and recurrent lesions were NR2F1-high . This pattern is consistent with NR2F1's role in cell cycle regulation, as evidenced by primary tumor tissues showing 67% Ki67-positive (proliferative) cells, none of which expressed NR2F1 .

What is the significance of NR2F1 in tumor dormancy research?

NR2F1 has been identified as a master regulator of tumor cell dormancy, making it a critical target for cancer research . When upregulated, NR2F1 induces expression of a dormancy gene signature, including transcription factors SOX9 and retinoic acid receptor β (RARβ), which in turn activate expression of cyclin-dependent kinase inhibitors p27 and p16, leading to G0/G1 cell cycle arrest and cell quiescence . Experimental evidence demonstrates that shRNA-mediated downregulation of NR2F1 results in reactivation of dormant tumor cells, leading to locoregional relapse and development of metastases . This functional relationship between NR2F1 and dormancy has been reproduced across various cancer models by multiple independent laboratories .

What are the molecular mechanisms through which NR2F1 regulates gene expression?

NR2F1 regulates gene expression through multiple mechanisms. First, it can bind directly to DNA as a dimer at direct repeat elements with the consensus sequence 5'-AGGTCA-3', as confirmed by binding studies . Second, NR2F1 can function by recruiting coactivator or corepressor complexes to modulate transcription . Third, it can act indirectly by serving as a cofactor for other nuclear receptors, including interactions with the retinoic acid receptor that influence neurological traits . NR2F1 also plays a significant role in epigenetic regulation by interacting with chromatin-remodeling enzymes to mediate global histone modifications . Chromatin immunoprecipitation coupled with DNA sequencing (ChIP-seq) has demonstrated that NR2F1 binding sites are frequently co-bound by other transcription factors like TFAP2A in human neural crest cells .

What are the key considerations when designing immunofluorescence experiments with NR2F1 antibodies?

When designing immunofluorescence (IF) experiments with NR2F1 antibodies, researchers must carefully consider several critical factors. First, antibody concentration significantly impacts staining patterns—higher concentrations may result in cytoplasmic staining, while lower concentrations may primarily highlight nuclear aggregates . Testing multiple concentrations (e.g., 2 μg/ml, 1 μg/ml, and 0.4 μg/ml) is recommended to determine optimal conditions for visualizing both diffuse nuclear signals and nuclear aggregates .

Second, proper controls are essential: negative controls should include cells stained with either primary antibody alone, secondary antibody alone, or just DAPI to identify potential background and autofluorescence . Third, fixation methods can significantly impact NR2F1 staining patterns and should be standardized and validated . Fourth, when investigating co-localization with other transcription factors, be aware that while NR2F1 may show both diffuse nuclear and aggregate patterns, other factors (like TFAP2A) may only show diffuse distribution despite functional interaction .

How should researchers validate the specificity of NR2F1 antibodies in their experimental systems?

Validating NR2F1 antibody specificity requires a multi-faceted approach. First, researchers should test the antibody in both wild-type (WT) and NR2F1-knockout models (CRISPR/Cas9 engineered cells or null mice) to confirm signal specificity . Second, multiple detection methods should be employed, including immunofluorescence (IF), flow cytometry (FC), and Western blotting (WB) to cross-validate results .

Third, comparison across multiple antibodies from different vendors or different clones is essential—studies have shown that different NR2F1 antibodies can produce variable staining patterns, particularly regarding nucleolar localization . Fourth, testing across different cell types is important as antibody performance may vary based on NR2F1 expression levels and cellular context . Finally, technical validation of HRP-conjugated antibodies specifically should include assessment of binding affinity post-conjugation and efficiency of antibody labeling, as these parameters can affect performance .

What controls are essential when using HRP-conjugated NR2F1 antibodies?

When working with HRP-conjugated NR2F1 antibodies, several controls are essential for ensuring experimental validity. First, include a negative control using matched isotype control antibodies conjugated to HRP to assess non-specific binding and background signal. Second, implement a CRISPR/Cas9 NR2F1-knockout cell line as a biological negative control to verify antibody specificity—this is particularly important given reported issues with antibody specificity in NR2F1 research .

Third, include positive controls using cell lines with confirmed high NR2F1 expression (such as certain dormant cancer cell lines like D-HEp3) . Fourth, perform a titration series to determine optimal antibody concentration, as concentration significantly impacts staining patterns and signal-to-noise ratios . Fifth, for HRP-specific considerations, include controls to verify that the HRP conjugation hasn't compromised antibody performance—this can be done by comparing results with unconjugated primary antibody plus HRP-conjugated secondary antibody systems .

How do fixation and permeabilization methods affect NR2F1 antibody performance?

Fixation and permeabilization methods significantly impact NR2F1 antibody performance and can influence observed subcellular localization patterns. Studies have shown that the apparent nucleolar-like staining pattern observed with some NR2F1 antibodies may be dependent on fixation methods . Researchers should systematically compare different fixation protocols (e.g., paraformaldehyde, methanol, acetone) and concentrations to determine optimal conditions for their specific experimental system.

Permeabilization agents (such as Triton X-100, saponin, or digitonin) and their concentrations should also be optimized, as they can differentially expose nuclear antigens. The duration of fixation and permeabilization steps can also affect epitope accessibility—over-fixation may mask epitopes while under-fixation may compromise cellular architecture. When using HRP-conjugated antibodies specifically, researchers should verify that fixation methods don't inactivate the HRP enzyme, which could affect signal development in downstream applications.

What are the optimal sample preparation techniques for Western blotting with HRP-conjugated NR2F1 antibodies?

For Western blotting with HRP-conjugated NR2F1 antibodies, sample preparation requires careful optimization. First, nuclear extraction protocols should be employed since NR2F1 is predominantly nuclear—commercial nuclear extraction kits or established protocols using hypotonic/high-salt buffers with protease inhibitors are recommended to preserve protein integrity. Second, protein estimation using Bradford or BCA assays should be performed to ensure equal loading.

Third, samples should be denatured at lower temperatures (70°C rather than 95°C) for shorter periods (5-7 minutes) to minimize potential epitope destruction, especially important for transcription factors. Fourth, freshly prepared samples generally yield better results than frozen-thawed lysates. Fifth, the addition of phosphatase inhibitors to lysis buffers is recommended when studying NR2F1 phosphorylation states. Sixth, for HRP-conjugated antibodies specifically, reducing agents in sample buffers should be carefully controlled, as they might affect HRP activity if samples are not adequately separated from the antibody during the procedure.

How can ChIP-seq be optimized for studying NR2F1 genomic binding sites?

Optimizing ChIP-seq for NR2F1 requires several specialized considerations. First, antibody selection is critical—the widely used H8132 clone has been successfully employed in previous ChIP-seq studies of NR2F1 , though researchers should validate this or other antibodies in their specific cell types. Second, crosslinking conditions should be optimized for transcription factors like NR2F1—a dual crosslinking approach using both formaldehyde (1%) and a protein-protein crosslinker like disuccinimidyl glutarate (DSG) can enhance recovery of transcription factor complexes.

Third, sonication conditions must be carefully calibrated to generate DNA fragments in the 200-500bp range without overheating samples, which could degrade the protein of interest. Fourth, implement a pre-clearing step with protein A/G beads to reduce background. Fifth, include appropriate controls such as Input DNA and IgG ChIP samples. Sixth, when analyzing data, focus on identifying motifs matching the known NR2F1 binding sequence (5'-AGGTCA-3') and investigate co-occupancy with known NR2F1 partners such as TFAP2A . Finally, validate key findings using orthogonal methods such as ChIP-qPCR.

What techniques are available to study NR2F1 protein-protein interactions?

Several sophisticated techniques can be employed to study NR2F1 protein-protein interactions. First, co-immunoprecipitation (Co-IP) followed by mass spectrometry represents a powerful approach to identify novel NR2F1 binding partners in an unbiased manner. Second, proximity ligation assay (PLA) enables visualization of protein interactions in situ with high sensitivity and specificity—particularly useful for confirming interactions between NR2F1 and suspected partners like RARβ or SOX9 .

Third, bimolecular fluorescence complementation (BiFC) can be used to visualize NR2F1 interactions in living cells. Fourth, FRET (Förster Resonance Energy Transfer) or FLIM (Fluorescence Lifetime Imaging Microscopy) provides spatial and temporal information about NR2F1 interactions with high resolution. Fifth, yeast two-hybrid or mammalian two-hybrid assays can map specific domains involved in protein-protein interactions. Finally, for HRP-conjugated antibodies specifically, hydrogen peroxide-mediated proximity labeling can be adapted to study proteins in close proximity to NR2F1 in fixed cells or tissues.

How can researchers differentiate between the various cellular pools of NR2F1 protein?

Differentiating between cellular pools of NR2F1 protein requires combining multiple advanced techniques. First, subcellular fractionation protocols can physically separate nuclear, cytoplasmic, nucleolar, and chromatin-bound fractions, followed by Western blotting to quantify NR2F1 in each compartment. Second, immunofluorescence microscopy with co-staining for compartment-specific markers is essential—nuclear NR2F1 can be distinguished using DAPI counterstaining, while nucleolar localization requires co-staining with nucleolar markers like fibrillarin or nucleolin .

Third, super-resolution microscopy techniques (STORM, STED, or PALM) provide nanoscale resolution to precisely localize NR2F1 within nuclear substructures. Fourth, biochemical approaches like salt extraction can separate loosely bound from tightly chromatin-associated NR2F1. Fifth, FRAP (Fluorescence Recovery After Photobleaching) with NR2F1-GFP fusion proteins can distinguish between mobile and immobile pools. Finally, pulse-chase experiments using protein synthesis inhibitors can differentiate newly synthesized from older NR2F1 protein pools, providing insights into protein turnover dynamics.

What approaches can be used to study NR2F1-mediated epigenetic modifications?

Studying NR2F1-mediated epigenetic modifications requires integrating several specialized techniques. First, ChIP-seq for histone modifications (H3K4me3, H3K27ac, H3K27me3, etc.) paired with NR2F1 ChIP-seq can reveal the epigenetic landscape at NR2F1 binding sites . Second, ChIP-reChIP (sequential ChIP) can determine whether NR2F1 and specific histone modifications co-occur on the same DNA molecules. Third, CUT&RUN or CUT&Tag provides higher resolution for mapping epigenetic modifications than traditional ChIP-seq.

Fourth, RNA-seq following NR2F1 manipulation (overexpression, knockdown, or knockout) can connect epigenetic changes to transcriptional outcomes. Fifth, ATAC-seq can reveal how NR2F1 influences chromatin accessibility. Sixth, Co-IP between NR2F1 and chromatin modifiers (like histone deacetylases or methyltransferases) can identify direct protein interactions. Finally, targeted epigenetic editing using CRISPR-dCas9 fused to epigenetic modifiers at NR2F1 binding sites can establish causality between specific modifications and gene expression changes.

How can researchers investigate the role of NR2F1 in tumor dormancy mechanisms?

Investigating NR2F1's role in tumor dormancy requires multifaceted approaches combining in vitro and in vivo techniques. First, 3D culture systems such as those using Matrigel represent valuable platforms—research has shown that T-HEp3 cells plated at low density in Matrigel and treated with NR2F1 agonists exhibit increased nuclear NR2F1 expression and dormancy phenotypes . Second, patient-derived xenograft (PDX) models have been instrumental in studying NR2F1-mediated dormancy, demonstrating that NR2F1 is upregulated in dormant residual cells .

Third, lineage tracing of disseminated tumor cells (DTCs) in animal models can monitor dormancy states over time. Fourth, single-cell RNA-seq of DTCs can reveal dormancy signatures and heterogeneity. Fifth, in vivo imaging using luciferase-tagged cells can track dormant cell populations non-invasively. Sixth, pharmacological approaches using NR2F1 agonists like compound C26 have shown promise in inducing dormancy programs and preventing metastasis . Finally, multiplexed immunohistochemistry for dormancy markers (NR2F1high/p27high/Ki-67low/p-S6low) in clinical samples can translate findings to human disease .

How should researchers interpret conflicting NR2F1 antibody staining patterns?

When encountering conflicting NR2F1 antibody staining patterns, researchers should implement a systematic investigation. First, compare multiple antibodies from different vendors and clones—studies have shown that different NR2F1 antibodies can produce variable staining patterns, particularly regarding nucleolar localization . Second, verify antibody specificity using CRISPR/Cas9 NR2F1-knockout controls—research has demonstrated that some nucleolar-like staining patterns observed with certain monoclonal antibodies may be unspecific .

Third, assess whether staining patterns are influenced by technical factors like fixation methods, antibody concentration, or cell type—evidence suggests that nucleolar-like patterns may depend on these variables . Fourth, employ multiple detection methods (IF, WB, FC) to cross-validate findings . Fifth, consider biological context—NR2F1 localization may genuinely differ between cell types or conditions. Finally, consult published literature critically, recognizing that historical observations of nucleolar NR2F1 localization may need reevaluation in light of more recent validation studies .

What are the common causes of high background when using HRP-conjugated NR2F1 antibodies?

High background when using HRP-conjugated NR2F1 antibodies can stem from several sources, each requiring specific troubleshooting approaches. First, excessive antibody concentration is a common cause—titration experiments with decreasing concentrations (e.g., 2 μg/ml to 0.4 μg/ml) should be performed to determine optimal signal-to-noise ratios . Second, insufficient blocking can lead to non-specific binding—extending blocking time and using alternative blocking agents (BSA, normal serum, commercial blockers) may improve results.

Third, endogenous peroxidase activity can generate background—implement a peroxidase quenching step (e.g., 3% H₂O₂ for 10 minutes) before antibody application. Fourth, cross-reactivity with similar epitopes may occur—review database information on the antibody's specificity profile and validation testing . Fifth, overly harsh fixation can increase autofluorescence and non-specific binding—optimize fixative concentration and duration. Finally, for HRP-conjugated antibodies specifically, storage conditions are critical—repeated freeze-thaw cycles or improper temperature storage can lead to increased non-specific binding as conjugates degrade.

What strategies can resolve inconsistencies between NR2F1 protein detection by Western blot versus immunofluorescence?

Resolving inconsistencies between Western blot and immunofluorescence requires systematic investigation of both technical and biological factors. First, epitope accessibility differs between methods—Western blotting detects denatured epitopes while IF detects native conformations, so antibody performance can vary dramatically between applications . Second, protein extraction methods for Western blotting may not efficiently capture all NR2F1 pools—optimize nuclear extraction protocols and compare with whole-cell lysates.

Third, post-translational modifications may affect antibody recognition—phosphatase or deglycosylation treatments prior to Western blotting may reveal whether modifications impact detection. Fourth, protein concentration thresholds differ between methods—Western blotting may detect bulk changes while IF can reveal cell-to-cell heterogeneity in NR2F1 expression . Fifth, for HRP-conjugated antibodies specifically, the conjugation might differentially impact performance in different applications—compare with unconjugated antibodies followed by secondary detection to isolate the variable. Finally, validate key findings using orthogonal approaches such as mass spectrometry or RT-qPCR to confirm protein presence and abundance.

How can researchers address the challenge of NR2F1 detection in tissues with naturally low expression?

Detecting NR2F1 in tissues with naturally low expression requires specialized amplification and sensitivity-enhancing techniques. First, implement tyramide signal amplification (TSA) when using HRP-conjugated antibodies—this catalytic reporter deposition technique can increase sensitivity by 10-100 fold. Second, employ antigen retrieval optimization—test multiple methods (heat-induced, enzymatic, pH variations) to maximize epitope exposure in fixed tissues. Third, extend primary antibody incubation time (overnight at 4°C or longer) to enhance binding to low-abundance targets.

Fourth, utilize signal enhancement systems like avidin-biotin complexes in conjunction with HRP detection. Fifth, consider concentration methods for Western blotting—immunoprecipitate NR2F1 before blotting to enrich the target protein. Sixth, explore alternative detection platforms like ultrasensitive Wes™ or Jess™ automated Western systems that can detect proteins at picogram levels. Finally, implement RNAscope® or other in situ hybridization techniques as complementary approaches to verify NR2F1 expression at the mRNA level when protein detection proves challenging.

What considerations are important when quantifying NR2F1 expression in heterogeneous tumor samples?

Quantifying NR2F1 expression in heterogeneous tumor samples requires specialized approaches to account for cellular diversity and spatial heterogeneity. First, implement multiplexed immunohistochemistry or immunofluorescence to simultaneously detect NR2F1 alongside cell type-specific markers and proliferation markers like Ki67—studies have shown NR2F1 expression is negatively correlated with Ki67 in tumor tissues . Second, utilize digital pathology with machine learning algorithms for unbiased quantification of staining intensity and distribution across tumor regions.

Third, employ laser capture microdissection to isolate specific regions for subsequent protein or RNA analysis. Fourth, implement single-cell approaches (mass cytometry, single-cell RNA-seq) to resolve cell-to-cell variation in NR2F1 expression. Fifth, establish clear quantification parameters—research has used stringent masks for strong nuclear NR2F1 signal appearing as prominent clusters to identify truly NR2F1-positive cells . Finally, report comprehensive metrics beyond simple "positive/negative" designations—include intensity distributions, percentage of positive cells, and spatial relationships with other markers to fully characterize heterogeneous expression patterns.

How might single-cell technologies advance our understanding of NR2F1 in heterogeneous cell populations?

Single-cell technologies hold tremendous promise for elucidating NR2F1's role in heterogeneous cell populations. First, single-cell RNA sequencing can reveal transcriptional states associated with varying NR2F1 expression levels, potentially identifying novel NR2F1-regulated genes and cellular subpopulations with distinct dormancy phenotypes. Second, single-cell ATAC-seq can map chromatin accessibility changes mediated by NR2F1, providing insights into its epigenetic regulatory functions at unprecedented resolution. Third, CyTOF (mass cytometry) with metal-conjugated NR2F1 antibodies can simultaneously quantify multiple protein markers alongside NR2F1 in thousands of individual cells.

Fourth, spatial transcriptomics technologies can preserve tissue context while providing single-cell resolution of NR2F1 expression patterns, particularly valuable for understanding tumor-microenvironment interactions in dormancy. Fifth, multiplexed ion beam imaging (MIBI) or imaging mass cytometry can visualize dozens of proteins simultaneously with subcellular resolution, enabling detailed characterization of NR2F1-positive cells in their native environment. These approaches collectively promise to resolve current contradictions in NR2F1 biology and reveal how cellular heterogeneity impacts dormancy and disease progression.

What are the emerging applications of NR2F1 agonists in cancer research?

NR2F1 agonists represent an exciting frontier in cancer research with multiple emerging applications. First, as demonstrated with compound C26, NR2F1 agonists can be used as experimental tools to induce and study dormancy mechanisms—activation of NR2F1 by C26 leads to a self-regulated increase in NR2F1 mRNA and protein and downstream transcription of a novel dormancy program . Second, these agonists show therapeutic potential for metastasis prevention—in preclinical models, agonist treatment resulted in inhibition of lung HNSCC metastasis, with disseminated tumor cells displaying a dormant NR2F1high/p27high/Ki-67low/p-S6low phenotype .

Third, NR2F1 agonists may serve as adjuvant therapy following primary tumor resection to suppress minimal residual disease. Fourth, combination strategies pairing NR2F1 agonists with epigenetic modifiers like 5-azacytidine could enhance efficacy by addressing the epigenetic silencing of NR2F1 observed in many cancers . Fifth, NR2F1 agonists might function as chemosensitizers by pushing rapidly dividing cells into a quiescent state before cytotoxic therapy. Finally, these compounds could potentially be used as molecular probes to identify patients with competent dormancy pathways who might benefit from dormancy-inducing therapeutic strategies.

How might differential antibody performance impact the interpretation of NR2F1 localization studies?

Differential antibody performance has profound implications for interpreting NR2F1 localization studies, requiring careful consideration of several factors. First, antibody clone-specific artifacts can lead to misinterpretation of subcellular localization—recent research has demonstrated that nucleolar-like staining patterns observed with certain monoclonal antibodies could not be confirmed with other antibodies and may represent nonspecific binding . Second, fixation-dependent epitope accessibility can dramatically alter apparent localization patterns—the same antibody may produce different staining patterns depending on fixation protocols .

Third, expression level-dependent antibody performance may create artifacts—staining patterns may vary with NR2F1 abundance rather than reflecting true biological differences in localization . Fourth, cell type-specific factors could influence antibody binding and apparent localization . Fifth, the influence of post-translational modifications on epitope accessibility may create discrepancies between studies using different antibodies. These considerations underscore the critical importance of rigorous validation using multiple antibodies, CRISPR/Cas9 knockout controls, and complementary techniques when studying NR2F1 localization.

What novel approaches could resolve current contradictions in NR2F1 localization patterns?

Resolving contradictions in NR2F1 localization patterns requires innovative approaches beyond conventional techniques. First, endogenous tagging of NR2F1 using CRISPR/Cas9 knock-in strategies would allow visualization of NR2F1 without reliance on antibodies, potentially resolving conflicting antibody-based observations . Second, live-cell imaging with fluorescently tagged NR2F1 could track dynamic localization changes in real-time, providing temporal information missing from fixed-cell studies. Third, super-resolution microscopy techniques (STORM, STED, PALM) offer nanoscale resolution that could definitively resolve subnuclear localization patterns.

Fourth, proximity labeling approaches like BioID or APEX could map the protein neighborhood of NR2F1 in different nuclear compartments. Fifth, correlative light and electron microscopy (CLEM) could provide ultrastructural context for fluorescence observations. Sixth, optogenetic tools to manipulate NR2F1 localization could establish causal relationships between localization and function. Finally, computational approaches integrating data from multiple antibodies and techniques could identify consistent patterns amidst technical variations, potentially revealing the true biological complexity of NR2F1 localization dynamics.

How will advances in structural biology influence our understanding of NR2F1 antibody interactions?

Advances in structural biology promise to revolutionize our understanding of NR2F1 antibody interactions through several avenues. First, high-resolution crystal or cryo-EM structures of NR2F1 in complex with antibodies could precisely map epitopes and explain differential antibody performance . Second, hydrogen-deuterium exchange mass spectrometry (HDX-MS) could identify NR2F1 regions with conformational flexibility, potentially explaining context-dependent antibody binding. Third, computational modeling combining antibody-antigen docking with molecular dynamics simulations could predict how post-translational modifications affect epitope accessibility.

Fourth, structural analysis of the NR2F1 ligand-binding domain (LBD) in various conformational states could explain how agonists like C26 induce structural changes that promote self-regulation . Fifth, integration of structural data with functional genomics could connect structural features to biological outcomes. Finally, structure-guided antibody engineering could produce next-generation NR2F1 antibodies with enhanced specificity for particular conformations or modifications, enabling more precise studies of NR2F1 biology and potentially resolving current contradictions in the literature.