BANF1 Antibody, FITC conjugated

What is BANF1 and what are its primary functions in cellular biology?

BANF1 (Barrier to Autointegration Factor 1) is a small DNA-binding protein involved in multiple cellular processes including nuclear assembly, chromatin organization, and regulation of gene expression. Most notably, BANF1 functions as a natural opponent of cGAS activity on genomic self-DNA, playing a key role in preventing inappropriate immune activation against self-DNA . The protein consists of 89-90 amino acids, with commercially available antibodies typically targeting regions such as amino acids 2-89, 1-89, 1-90, or specific terminal regions .

BANF1 has emerged as a significant factor in cancer biology, with research showing that upregulated expression in tumor tissues is significantly associated with poor survival outcomes and is negatively correlated with immune cell infiltration . Studies have demonstrated that BANF1 knockout can antagonize tumor growth in immunocompetent mice, suggesting its importance in tumor progression and immune evasion mechanisms .

Why would researchers choose a FITC-conjugated BANF1 antibody over unconjugated alternatives?

FITC-conjugated BANF1 antibodies, such as ABIN7145260, offer several methodological advantages for researchers. The direct fluorescent conjugation eliminates the need for secondary antibody incubation steps in immunofluorescence (IF) protocols, reducing background signal and potential cross-reactivity issues . This direct detection approach is particularly valuable for multicolor immunofluorescence experiments where researchers need to simultaneously visualize multiple targets using different fluorophores.

FITC (fluorescein isothiocyanate) has an excitation maximum around 495 nm and emission maximum around 519 nm, making it compatible with standard fluorescence microscopy setups and flow cytometry instruments. For researchers investigating BANF1's subcellular localization or studying its expression in tissues and cell populations, FITC-conjugated antibodies provide a direct visualization method without requiring additional reagents or amplification steps .

What epitopes are targeted by commercially available BANF1 antibodies?

Commercial BANF1 antibodies target various epitopes across the protein, providing researchers with options depending on their experimental needs. Based on the available product information, several regions are commonly targeted:

The specific epitope can be critical depending on research objectives - for example, antibodies targeting the N-terminal region may be preferable when studying interactions with other proteins, while those targeting specific amino acid sequences may offer higher specificity for certain applications .

What is the species reactivity spectrum of available BANF1 antibodies?

The species reactivity of BANF1 antibodies varies across products, with human reactivity being most common. The FITC-conjugated BANF1 antibody (ABIN7145260) specifically targets human BANF1 . Other commercially available BANF1 antibodies demonstrate different reactivity profiles:

For researchers working with mouse or rat models, antibodies with cross-reactivity to these species would be necessary, such as the N-terminal targeting antibody ABIN499409, which reacts with human, mouse, and rat BANF1 . This cross-species reactivity is particularly valuable for translational research comparing BANF1 function across different mammalian models .

What are the optimal applications for FITC-conjugated BANF1 antibodies?

FITC-conjugated BANF1 antibodies excel in several experimental applications, with immunofluorescence microscopy being the primary application. These antibodies are particularly well-suited for:

• Cell localization studies: Visualizing BANF1's nuclear distribution and potential redistribution under various cellular conditions or treatments.

• Flow cytometry: Quantifying BANF1 expression levels across cell populations, especially useful when studying cancer cell heterogeneity.

• Multicolor immunofluorescence: Combining with other antibodies carrying different fluorophores to study co-localization with interaction partners.

The FITC-conjugated BANF1 antibody (ABIN7145260) is purified using Protein G affinity chromatography (>95% purity) and targets amino acids 2-89 of human BANF1 . When designing experiments, researchers should consider that direct conjugation may result in lower signal intensity compared to amplification methods using secondary antibodies, though with improved specificity and reduced background .

How do experimental outcomes differ between polyclonal and monoclonal BANF1 antibodies?

The choice between polyclonal and monoclonal BANF1 antibodies significantly impacts experimental results:

Polyclonal antibodies like the FITC-conjugated ABIN7145260 may detect BANF1 even if some epitopes are masked or modified, making them valuable for detecting BANF1 across different experimental conditions . Monoclonal antibodies offer superior reproducibility and specificity, which is crucial for quantitative studies comparing BANF1 expression across samples .

What controls should be incorporated when using BANF1 antibodies in immunofluorescence studies?

Robust experimental design for BANF1 immunofluorescence studies requires several critical controls:

• Positive control: Use cell lines or tissues known to express BANF1, such as cancer cell lines from colon adenocarcinoma (COAD), lung adenocarcinoma (LUAD), or melanoma (SKCM), which have been shown to exhibit high BANF1 expression .

• Negative control: Include samples where BANF1 expression is eliminated through CRISPR-Cas9 knockout, as described in the B16F10 and MC38 cell line models .

• Isotype control: Use a non-specific antibody of the same isotype (IgG for ABIN7145260) and same conjugate (FITC) to assess non-specific binding .

• Absorption control: Pre-incubate the FITC-conjugated BANF1 antibody with recombinant BANF1 protein before staining to confirm specificity.

• Secondary-only control: For comparison with indirect detection methods, include samples treated only with secondary antibody.

These controls are essential for distinguishing specific BANF1 staining from autofluorescence or non-specific binding, particularly important when studying BANF1's role in complex tissue microenvironments such as tumors .

How can BANF1 antibodies be utilized to study the cGAS-STING pathway in cancer immunity?

BANF1 antibodies, including FITC-conjugated variants, serve as critical tools for investigating the cGAS-STING pathway and its relationship to cancer immunity. Research has established BANF1 as a natural opponent of cGAS activity on genomic self-DNA, making it a key regulator of this pathway . Methodological approaches include:

• Co-localization studies: Using FITC-conjugated BANF1 antibodies in combination with antibodies against cGAS to visualize their spatial relationship in the nucleus and cytoplasm under various cellular stress conditions.

• Pathway activation analysis: Measuring cGAS-STING pathway activation markers (phospho-STING, IFN-β, inflammatory cytokines) in BANF1 knockout versus wild-type cells to quantify BANF1's suppressive effect.

• Chromatin association dynamics: Tracking BANF1's association with chromatin before and after DNA damage to understand how it shields genomic DNA from cGAS recognition.

Recent research demonstrates that BANF1 knockout activates antitumor immune responses mediated by the cGAS-STING pathway, resulting in an immune-activating tumor microenvironment with increased CD8+ T cell infiltration and decreased myeloid-derived suppressor cell enrichment . This suggests that BANF1 functionally suppresses cGAS-STING signaling in tumor cells, providing a potential mechanism for its association with poor prognosis in multiple cancer types .

What methodologies can reveal BANF1's impact on tumor microenvironment and immune cell infiltration?

Investigating BANF1's impact on the tumor microenvironment requires sophisticated methodological approaches:

• Comparative immunohistochemistry: Using BANF1 antibodies on tissue microarrays to correlate BANF1 expression with immune cell markers. Research has shown that BANF1 expression is negatively correlated with immune cell infiltration in 15 of 33 TCGA cancer types .

• Flow cytometric analysis: Combining surface marker staining with intracellular BANF1 detection to quantify immune cell populations in BANF1-high versus BANF1-low tumors.

• Single-cell RNA sequencing: Correlating BANF1 expression levels with immune cell signatures at single-cell resolution to understand cellular heterogeneity.

• Spatial transcriptomics: Mapping BANF1 expression patterns alongside immune cell distributions within the tumor microenvironment.

Research shows that BANF1 knockout reshapes the tumor microenvironment into a "hot" inflamed T cell-infiltrated tumor, with increased CD8+ T cell infiltration and decreased myeloid-derived suppressor cells . This remodeling appears mechanistically related to innate immune responses activated by the cGAS-STING pathway . These findings highlight the value of using BANF1 antibodies to profile expression patterns within heterogeneous tumor samples and correlate them with immune infiltration markers.

How can researchers assess the relationship between BANF1 expression and clinical outcomes in cancer patients?

Methodological approaches to evaluate BANF1's relationship with clinical outcomes include:

• Tissue microarray analysis: Using BANF1 antibodies for immunohistochemistry on cancer tissue arrays containing multiple tumor types. Studies have used this approach to examine BANF1 expression across 11 common tumor types including thyroid, esophageal, gastric, colon, rectal, liver, pancreatic, lung, breast, and kidney cancers .

• Survival analysis correlation: Correlating BANF1 expression levels with patient survival data. Research using TCGA data has shown that upregulated BANF1 expression is significantly associated with poor survival outcomes in multiple cancer types .

• Immune signature correlation: Using bioinformatics approaches like GSEA (Gene Set Enrichment Analysis) to correlate BANF1 expression with immune-related gene signatures. Low BANF1 expression has been associated with enriched immune signatures including inflammatory response and allograft rejection .

• Immunotherapy response prediction: Evaluating BANF1 expression as a potential biomarker for immunotherapy response. Clinical cohort data suggests patients with high BANF1 expression had worse prognosis following immunotherapy .

The ESTIMATE algorithm has been used to analyze immune cell infiltration in relation to BANF1 expression, revealing negative correlations between BANF1 and immune scores in multiple cancer types, including colon adenocarcinoma (pearson r=-0.14, p<0.02), lung adenocarcinoma (pearson r=-0.28, p<0.001), and skin cutaneous melanoma (pearson r=-0.16) .

How should researchers address potential false-positive or false-negative results when using FITC-conjugated BANF1 antibodies?

When working with FITC-conjugated BANF1 antibodies, researchers should implement several strategies to mitigate false results:

• Autofluorescence control: FITC emission overlaps with cellular autofluorescence, particularly in tissues like liver or kidney. Include unstained controls and consider autofluorescence quenching reagents.

• Fixation optimization: Test multiple fixation protocols, as overfixation may mask the BANF1 epitope while underfixation may alter BANF1's nuclear localization.

• Antibody validation: Validate antibody specificity using BANF1 knockout cells as negative controls. The literature describes successful CRISPR-Cas9 knockout of BANF1 in B16F10 and MC38 cell lines that could serve as validation tools .

• Orthogonal verification: Confirm FITC-conjugated antibody results with unconjugated BANF1 antibodies using indirect detection methods.

• Titration experiments: Perform antibody dilution series to determine optimal concentration that maximizes signal-to-noise ratio.

For accurate data interpretation, researchers should quantify BANF1 expression relative to carefully selected control samples and normalize for cell number and imaging parameters. When discrepancies arise, consider that direct FITC conjugation may reduce antibody sensitivity compared to signal amplification methods used with unconjugated primary antibodies .

What methodological considerations should guide the analysis of BANF1's role in tumor growth studies?

Research investigating BANF1's impact on tumor growth requires careful methodological considerations:

• Model selection: Studies show markedly different outcomes between immunocompetent and immunodeficient models. In C57BL/6 mice, BANF1 deficiency significantly inhibited tumor growth (20%–40% tumor formation in BANF1 KO vs 100% in control), while in nude mice, the effect was minimal .

• Genetic manipulation verification: Confirm BANF1 knockout efficiency through multiple methods (Western blot, qPCR, sequencing). Address potential off-target effects by including rescue experiments - research has shown that re-expression of sgRNA-resistant BANF1 reverses tumor growth inhibition in knockout models .

• Immune memory assessment: Consider rechallenge experiments to test immune memory development. Studies demonstrate that mice achieving tumor-free status after initial BANF1 KO tumor rejection could subsequently reject wild-type tumor cells .

• Mechanistic investigation: Include parallel experiments to assess cGAS-STING pathway activation, as BANF1 knockout has been shown to activate antitumor immunity through this mechanism .

• Combination therapy approaches: When evaluating BANF1 targeting in combination with immunotherapies like anti-PD-1, use appropriate dosing schedules and control groups to distinguish synergistic from additive effects .

Data interpretation should account for differences in tumor microenvironment composition between models, particularly focusing on CD8+ T cell infiltration and myeloid-derived suppressor cell populations, which have been shown to be significantly altered by BANF1 status .

How can researchers interpret BANF1 expression data across different cancer types?

Interpreting BANF1 expression across cancer types requires consideration of several factors:

• Baseline tissue variation: BANF1 expression varies naturally between tissue types. TCGA pan-cancer analysis shows higher BANF1 expression in 15 tumors compared to normal counterparts, including colon adenocarcinoma (COAD), lung adenocarcinoma (LUAD), and liver hepatocellular carcinoma (LIHC) .

• Correlation with immune signatures: BANF1 expression negatively correlates with immune cell infiltration in 15 of 33 TCGA cancer types, with stronger correlations in specific cancers:

  • Colon adenocarcinoma: pearson r=-0.14, p<0.02

  • Lung adenocarcinoma: pearson r=-0.28, p<0.001

  • Skin cutaneous melanoma: pearson r=-0.16, p<0.05

• Prognostic significance variation: While high BANF1 expression generally correlates with poor prognosis, the strength of this association varies by cancer type.

• Technical considerations: When comparing BANF1 expression data from different techniques (IHC, WB, RNA-seq), researchers should note that antibodies targeting different epitopes (N-term vs C-term vs full length) may yield different results .

To standardize interpretation, researchers should employ quantitative analysis methods such as H-score for IHC or normalized counts for RNA-seq, and validate findings across multiple patient cohorts when possible. The multiorgan tissue chip approach used in literature (containing 11 common tumor types with 2-6 cases each) provides a methodology for comparative analysis across cancer types .

How is BANF1 being investigated as a potential therapeutic target in cancer?

BANF1 has emerged as a promising therapeutic target in cancer based on several key findings:

• Selective tumor growth inhibition: BANF1 knockout antagonizes tumorigenesis in immunocompetent mice but shows minimal effect in immunocompromised models, suggesting its targeting could selectively inhibit tumors while activating anti-tumor immunity .

• Immunotherapy synergy: Combining BANF1 knockout with anti-PD-1 antibody treatment shows improved therapeutic benefit compared to anti-PD-1 alone in preclinical models, highlighting potential for combination therapy approaches .

• Immune memory induction: Mice that achieved tumor-free status after rejecting BANF1-knockout tumors could subsequently reject wild-type tumor rechallenge, indicating development of durable anti-tumor immune memory .

• Pathway-specific targeting: BANF1 inhibition activates the cGAS-STING pathway specifically in tumor cells, potentially offering a mechanism to convert "cold" tumors to "hot" immunologically active tumors .

Current research directions include developing small molecule inhibitors targeting BANF1's DNA-binding capacity, exploring BANF1-targeting antibody-drug conjugates, and investigating RNA interference approaches to downregulate BANF1 expression. Researchers are also exploring biomarker strategies to identify patients most likely to benefit from BANF1-targeting therapies, with focus on those showing BANF1 upregulation and poor immune cell infiltration .

What methodological approaches can assess BANF1's interaction with the cGAS-STING pathway?

Investigating BANF1's interaction with the cGAS-STING pathway requires sophisticated methodological approaches:

• Proximity ligation assays: Using BANF1 antibodies in combination with cGAS antibodies to visualize and quantify their molecular proximity in situ.

• Co-immunoprecipitation studies: Employing BANF1 antibodies for pull-down experiments followed by cGAS detection to assess physical interaction.

• STING pathway activity assessment: Measuring downstream markers including:

  • Phosphorylated STING

  • TBK1 activation

  • IRF3 nuclear translocation

  • Type I interferon production

  • Inflammatory cytokine expression

• Chromatin accessibility analysis: Using techniques like ATAC-seq to examine how BANF1 manipulation affects chromatin states and DNA accessibility to cGAS.

• Live cell imaging: Monitoring BANF1-DNA interactions using fluorescently tagged proteins to understand real-time dynamics of DNA shielding from cGAS.

Research has demonstrated that BANF1 knockout activates antitumor immune responses mediated by the cGAS-STING pathway . Gene set enrichment analysis (GSEA) of tumors with low BANF1 expression shows enrichment of immune-associated signatures, including inflammatory response, allograft rejection, and TNFA signaling via NFKB . These methodological approaches help elucidate the mechanistic relationship between BANF1 and innate immune sensing.

How might FITC-conjugated BANF1 antibodies contribute to understanding BANF1's role in immune checkpoint inhibitor response?

FITC-conjugated BANF1 antibodies offer unique capabilities for investigating BANF1's role in immune checkpoint inhibitor response:

• Multiparameter flow cytometry: Combining FITC-conjugated BANF1 antibodies with markers for T cell exhaustion, activation, and checkpoint expression to correlate BANF1 levels with immunotherapy-relevant phenotypes at single-cell resolution.

• Multiplex immunofluorescence imaging: Using FITC-BANF1 antibodies alongside markers for PD-1, PD-L1, and immune cell subsets to map spatial relationships within the tumor microenvironment before and after checkpoint inhibitor treatment.

• Ex vivo patient sample analysis: Assessing BANF1 expression in tumor biopsies from patients undergoing immune checkpoint inhibitor therapy to correlate baseline expression with treatment outcomes.

• Dynamic response monitoring: Tracking changes in BANF1 expression during immunotherapy treatment to identify potential adaptive resistance mechanisms.

Research has revealed that in immunotherapy clinical cohorts, patients with high BANF1 expression had worse prognosis, suggesting BANF1 as a potential biomarker for checkpoint inhibitor response . Further studies combining BANF1 knockout with anti-PD-1 treatment demonstrated improved therapeutic benefit compared to anti-PD-1 alone, highlighting BANF1's potential role in modulating response to checkpoint blockade .

These approaches using FITC-conjugated BANF1 antibodies could help identify patients most likely to benefit from combination strategies targeting both BANF1 and immune checkpoints, potentially overcoming resistance mechanisms in currently non-responsive tumors.