FOXI1 Antibody

What is FOXI1 and why is it significant in research?

FOXI1 is a forkhead transcription factor with a conserved DNA binding domain structurally similar to the winged-helix structures of histones H1 and H5. It plays crucial roles in chromatin remodeling and can create site-specific changes in chromatin structure. Unlike most transcription factors, FOXI1 remains bound to condensed chromosomes during mitosis, suggesting its importance in maintaining transcriptional "ground states" during cell division . FOXI1 is dynamically expressed in dividing cells of otic placode and pharyngeal pouches, and FOXI1-null mutants display highly specific phenotypes in zebra fish and mouse ears . Recent research has also implicated FOXI1 in pathways related to HPV life cycle regulation .

What species reactivity do FOXI1 antibodies typically exhibit?

Most commercially available FOXI1 antibodies are validated for reactivity with human and mouse samples . While not all antibodies are explicitly tested in other species, cross-reactivity may be possible with species that have high sequence homology. If you need to use FOXI1 antibody on non-validated species, running a BLAST analysis between your species of interest and the immunogen sequence can help predict potential cross-reactivity . For instance, researchers have successfully used human/mouse-reactive FOXI1 antibodies in IHC applications with mouse tissue and inquired about potential cross-reactivity with horse and pig tissues .

What are the common applications for FOXI1 antibodies?

FOXI1 antibodies have been validated for multiple experimental applications, including:

Each application requires specific optimization for FOXI1 detection .

How should cells be prepared for FOXI1 immunofluorescence detection?

For optimal FOXI1 immunofluorescence detection, cells should be fixed with 4% formaldehyde-0.1% Triton X-100 in phosphate-buffered saline at room temperature for 30 minutes . This fixation protocol preserves both nuclear and potential cytoplasmic FOXI1 localization while ensuring adequate permeabilization. After fixation, proceed with standard washing steps and incubation with your primary FOXI1 antibody (such as anti-GFP for GFP-tagged FOXI1 or anti-V5 for V5-tagged FOXI1 constructs) . For visualization, confocal microscopy provides optimal resolution of nuclear localization patterns. If studying FOXI1 during mitosis, synchronization protocols may be beneficial to enrich for mitotic cells where FOXI1's unique chromosome binding can be observed .

What are the recommended storage conditions for maintaining FOXI1 antibody stability?

For long-term storage (up to one year), store FOXI1 antibodies at -20°C . For short-term storage and frequent use, keep at 4°C for up to one month . When using FOXI1 antibodies conjugated with fluorophores (such as Janelia Fluor® 669), store at 4°C in the dark to prevent photobleaching . Avoid repeated freeze-thaw cycles as they can degrade antibody quality and compromise experimental results . If necessary, prepare working aliquots upon first thaw to minimize freeze-thaw cycles. For diluted working solutions, prepare fresh on the day of experiment or store at 4°C with preservative (such as 0.05% sodium azide) for short periods only .

How can I design a ChIP experiment to investigate FOXI1 binding to target DNA sequences?

To perform chromatin immunoprecipitation (ChIP) for FOXI1, follow this optimized protocol:

• Crosslinking: Fix cells with 1% formaldehyde for 10 minutes at room temperature to preserve DNA-protein interactions .

• Chromatin preparation: Lyse cells and sonicate chromatin to generate 200-500bp fragments.

• Immunoprecipitation: Incubate sonicated chromatin with FOXI1 antibody (5μg) overnight at 4°C .

• Washing: Perform stringent washes to remove non-specific binding.

• Elution and reversal: Elute FOXI1-DNA complexes with elution buffer (1% SDS, 0.1M NaHCO₃) and reverse crosslinking by heating at 65°C for 6 hours .

• DNA purification: Perform proteinase K digestion and phenol-chloroform extraction .

• Analysis: Analyze enriched DNA by qPCR, sequencing, or cloning into vectors for further characterization .

When designing primers for qPCR validation, divide your target region into segments of 150-300bp for optimal resolution of binding sites . In published studies, researchers have successfully identified FOXI1 binding by dividing the target regions into four segments (I-IV) and using specific primer pairs for each segment .

How can I quantitatively assess FOXI1-induced changes in chromatin accessibility?

To measure FOXI1-induced changes in chromatin accessibility, implement a quantitative DNase I hypersensitivity assay as follows:

• Cell preparation: Culture cells to confluence and maintain for 2-3 days before splitting 1:4 .

• Optional synchronization: For mitotic studies, add nocodazole (400 ng/ml) for 16-20 hours; confirm by observing condensed nuclei in 90% of shake-off cells .

• Nuclei isolation: Extract nuclei under conditions that preserve nuclear architecture.

• DNase I treatment: Treat nuclei with increasing concentrations of DNase I.

• DNA purification: Isolate DNA from treated samples.

• Quantitative assessment: Perform real-time PCR using primers targeting regions of interest .

• Data analysis: Calculate sensitivity as the ratio of signal in DNase I-treated versus untreated samples.

This method reveals that FOXI1 creates stable alterations in DNase I sensitivity that become more pronounced in condensed chromosomes . In most cases, FOXI1 expression increases resistance to DNase I, though specific target sites may become more accessible, indicating context-dependent chromatin remodeling activity .

What controls should be included when studying FOXI1 binding specificity in ChIP-qPCR experiments?

For rigorous ChIP-qPCR experiments investigating FOXI1 binding specificity, include these essential controls:

• Input control: Non-immunoprecipitated chromatin (typically 1-5% of starting material) to normalize for DNA abundance differences.

• IgG control: Non-specific IgG from the same species as the FOXI1 antibody to assess background binding.

• Positive control region: A genomic region with established FOXI1 binding (based on consensus sequence RYMAAYA) .

• Negative control region: A transcriptionally active region without predicted FOXI1 binding sites.

• Peptide competition: When possible, include blocked antibody controls using the immunizing peptide, as demonstrated in the paraffin-embedded human breast carcinoma tissue experiment .

• Multiple primer sets: Design primers covering several segments of your target region to precisely map binding locations .

• Cross-validation: Confirm results using multiple bioinformatics databases (JASPAR, HOCOMOCO, TRANSFAC) to identify consistent binding motifs .

In published studies, researchers successfully identified specific binding of transcription factors including FOXI1 by dividing target regions into segments and comparing enrichment patterns across these segments .

How does FOXI1 expression change during cell differentiation, and how should this influence experimental design?

FOXI1 expression significantly decreases during epithelial cell differentiation, which has important implications for experimental design. Immunohistochemistry studies on organotypic epithelial raft cultures show that FOXI1 nuclear staining is predominantly observed in all basal cells and only sparsely in cells of suprabasal layers . This differentiation-dependent expression pattern correlates with changes in DNA binding activity observed between differentiated and undifferentiated cells .

When designing experiments:

• Sample selection: Choose appropriate cell layers based on differentiation status; use markers to confirm differentiation stage.

• Timing considerations: For inducible systems, determine optimal induction timing relative to differentiation status.

• 3D models: Consider using organotypic raft cultures that recapitulate stratified epithelia rather than simple monolayer cultures.

• Comparative analysis: Always include both undifferentiated and differentiated samples for direct comparison.

• Multi-parameter analysis: Combine FOXI1 detection with differentiation markers to correlate expression patterns.

These considerations are particularly important when studying FOXI1's role in processes like HPV life cycle regulation, where viral activity varies across epithelial differentiation stages .

What are potential causes for inconsistent FOXI1 antibody performance across different experimental conditions?

When encountering variable results with FOXI1 antibodies, consider these potential factors:

When troubleshooting, remember that some lots of FOXI1 antibodies (e.g., A07512-1) are available in BSA-free formulations, which may be required for certain applications . Additionally, for cross-species applications not explicitly validated (e.g., pig, horse), consider running pilot tests after sequence homology analysis .

How can I validate that my FOXI1 antibody is detecting the correct protein in Western blot applications?

To rigorously validate FOXI1 antibody specificity in Western blot applications:

• Molecular weight verification: Confirm that the detected band matches FOXI1's expected molecular weight of approximately 41 kDa .

• Positive control: Include lysate from cells known to express FOXI1 (e.g., specific kidney or breast tissue samples) .

• Negative control: Use lysate from cell lines with confirmed low/no FOXI1 expression or FOXI1 knockout cells.

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding; this should eliminate true FOXI1 bands .

• siRNA/shRNA validation: Compare samples with and without FOXI1 knockdown to confirm signal reduction.

• Recombinant protein: Run purified or overexpressed FOXI1 protein as reference.

• Multiple antibodies: When possible, verify with a second FOXI1 antibody targeting a different epitope.

• Tagged constructs: For overexpression studies, compare detection with both FOXI1 antibody and tag-specific antibody (e.g., GFP or V5 for fusion proteins) .

When submitting results for evaluation, include detailed protocol information, lot numbers, and tissue sources as demonstrated in the customer Q&A regarding colon and kidney samples .

What considerations are important when interpreting FOXI1 localization data from immunofluorescence experiments?

When interpreting FOXI1 localization in immunofluorescence experiments, consider these critical factors:

• Nuclear vs. cytoplasmic signal: FOXI1 shows intense nuclear staining in basal layer cells, but may also display cytoplasmic localization in certain cell types . Evaluate both compartments separately.

• Cell cycle stage: Unlike most transcription factors, FOXI1 remains bound to condensed chromosomes during mitosis . Document cell cycle phase when analyzing localization patterns.

• Differentiation status: FOXI1 expression gradually decreases from basal to superficial layers in stratified epithelia . Note the differentiation state of cells when interpreting localization patterns.

• Co-localization studies: Consider dual staining with chromatin markers or other transcription factors to understand functional interactions.

• Fixation artifacts: Different fixation methods may affect apparent localization; validate with multiple protocols.

• Antibody specificity: Include peptide competition controls to distinguish specific from non-specific staining .

• Signal intensity gradients: Quantify nuclear signal intensity across different cell populations rather than making binary assessments.

Proper interpretation requires understanding that FOXI1's localization is dynamic and context-dependent, particularly in tissues with differentiation gradients like stratified epithelia .

How is FOXI1 implicated in human papillomavirus (HPV) life cycle regulation?

Recent research has revealed FOXI1's involvement in HPV life cycle regulation, particularly in relation to the HPV-18 genome. Although ChIP-qPCR analyses did not show significant direct binding of FOXI1 to segments of the HPV-18 Long Control Region (LCR) compared to other transcription factors like PAX6, HMGB1, and NFE2 , functional studies demonstrate that FOXI1 impacts HPV-18 P105 transcriptional activity .

FOXI1 expression decreases upon differentiation in organotypic epithelial raft cultures, with immunohistochemistry showing nuclear staining in all basal cells but only sparse staining in suprabasal layers . This differentiation-dependent expression pattern correlates with HPV life cycle progression, which requires coordinated expression of viral genes as epithelial cells differentiate.

These findings suggest FOXI1 may contribute to HPV life cycle regulation through indirect mechanisms or through interactions with other transcription factors, potentially by modifying chromatin accessibility in HPV-infected cells. Further research is needed to fully characterize FOXI1's role in viral transcriptional regulation and its potential as a target for antiviral strategies .

What is known about FOXI1's role in chromatin remodeling and how does this affect experimental approaches?

FOXI1 demonstrates unique capabilities in chromatin remodeling that distinguish it from most transcription factors. Key findings include:

• Mitotic chromosome binding: Unlike most transcription factors that dissociate during mitosis, FOXI1 remains bound to condensed chromosomes throughout cell division , suggesting a role in preserving transcriptional states across cell generations.

• DNase I sensitivity modulation: FOXI1 creates stable alterations in DNase I sensitivity that become more pronounced in condensed chromosomes . The majority of chromatin regions show increased resistance to DNase I in FOXI1-expressing cells, though some sites become more accessible .

• Nucleosomal structure modification: Micrococcal nuclease partial digestion generates oligonucleosome ladders that are slightly longer in FOXI1-expressing cells compared to controls, indicating changes in nucleosome spacing or organization .

• Consensus sequence recognition: ChIP studies show that 88% of FOXI1-bound DNA fragments contain the consensus binding sites for Fox proteins .

These properties suggest FOXI1 functions as a "pioneer factor" that can access condensed chromatin and create stable structural changes. When designing experiments, researchers should:

• Consider cell cycle synchronization to study FOXI1's mitosis-specific functions

• Implement chromatin accessibility assays alongside expression studies

• Extend analyses beyond typical transcriptional readouts to include structural chromatin changes

• Include controls for both active and repressive chromatin states

This chromatin remodeling capability may explain FOXI1's role in establishing transcriptional "ground states" during development and tissue differentiation .

What methodological advances have improved the study of FOXI1 binding and function?

Recent methodological advances have significantly enhanced our ability to study FOXI1 binding and function:

• Inducible expression systems: Development of stable cell lines expressing FoxI1-GFP or FoxI1-V5 fusion proteins under control of doxycycline-inducible systems allows precise temporal control of FOXI1 expression .

• Live cell imaging: Fusion with fluorescent proteins like GFP enables direct visualization of FOXI1 dynamics throughout the cell cycle in living cells .

• ChIP-sequencing integration: Combining chromatin immunoprecipitation with high-throughput sequencing provides genome-wide maps of FOXI1 binding sites .

• Integrative bioinformatics: Multi-database approaches (JASPAR, HOCOMOCO, TRANSFAC) to identify consistent binding motifs improve prediction accuracy of true binding sites .

• Quantitative DNase I hypersensitivity assays: Real-time PCR-based methods enable precise quantification of FOXI1-induced changes in chromatin accessibility .

• Organotypic 3D cultures: These models better recapitulate the differentiation gradients in which FOXI1 functions compared to monolayer cultures .

• Fluorophore conjugation: Direct conjugation with advanced fluorophores like Janelia Fluor® 669 improves detection sensitivity in applications like flow cytometry and immunofluorescence .

• BSA-free antibody formulations: Custom preparations without bovine serum albumin reduce background and cross-reactivity in sensitive applications .

These methodological improvements allow researchers to better understand FOXI1's dynamic binding properties and its context-dependent functions in different cellular environments and developmental stages.