FOXO6 Antibody, HRP conjugated

What is FOXO6 and why is it significant in research?

FOXO6 is a member of the forkhead box family of transcription factors that functions as a transcriptional activator . Research has shown that FOXO6 plays critical roles in multiple biological contexts including cancer progression, glycolysis regulation, cardiac remodeling, and craniofacial development . It has high expression in hepatocellular carcinoma tissues, and its knockdown has been shown to inhibit glycolysis and chemotherapy resistance through the PI3K/Akt signaling pathway . FOXO6 has also been implicated in cardiac pathological remodeling through activation of the Kif15/TGF-β1 axis . Its developmental expression is particularly notable in craniofacial tissues, where it regulates Hippo signaling and post-natal growth .

What are the differences between unconjugated and HRP-conjugated FOXO6 antibodies?

Unconjugated FOXO6 antibodies require a secondary detection system while HRP-conjugated variants have horseradish peroxidase directly attached to the antibody molecule, enabling direct detection . The HRP-conjugated antibodies are ideal for applications where signal amplification is needed without multiple incubation steps, particularly useful in ELISA, Western blot, and certain immunohistochemistry protocols . The conjugation provides enhanced sensitivity and reduces background compared to two-step detection systems, although the molecular weight of the antibody is increased by the addition of the HRP enzyme . Storage requirements are generally more stringent for conjugated antibodies, typically at -20°C or -80°C to maintain enzyme activity .

How should researchers choose between polyclonal, monoclonal and recombinant FOXO6 antibodies?

The choice depends on the specific research application and requirements:

For FOXO6, recombinant antibodies (like product 83926-1-RR) have demonstrated excellent performance in multiple applications including Western blot, IHC, and IF/ICC with consistent results across different experimental conditions .

What are the optimal conditions for using FOXO6 antibody in Western blot applications?

For optimal Western blot results using FOXO6 antibodies, researchers should consider several critical parameters:

• Sample preparation: Protein extraction should include protease inhibitors and phosphatase inhibitors if studying phosphorylated forms of FOXO6.

• Dilution optimization:

  • For unconjugated antibodies: 1:500-1:6000 dilution range, with 1:1000 being a recommended starting point

  • For HRP-conjugated antibodies: Generally higher dilutions (1:1000-1:10000) are effective

• Detection considerations: When using HRP-conjugated FOXO6 antibodies, researchers should note the expected molecular weight (calculated as 51 kDa, but typically observed at 70-80 kDa range) . This discrepancy is consistent across antibodies from different manufacturers and likely reflects post-translational modifications.

• Validation controls: Positive controls should include samples known to express FOXO6, such as HeLa cells, HepG2 cells, C2C12 cells, mouse brain tissue, or mouse lung tissue .

How can FOXO6 antibodies be effectively applied in immunohistochemistry and immunofluorescence studies?

Effective application of FOXO6 antibodies in IHC and IF requires the following methodological considerations:

• Antigen retrieval: For optimal results, use TE buffer at pH 9.0 for antigen retrieval, although citrate buffer at pH 6.0 can serve as an alternative . This step is critical as formalin fixation can mask FOXO6 epitopes.

• Recommended dilutions:

  • For IHC: 1:200-1:1000, with validation in rat brain tissue, human gliomas tissue, and human brain tissue

  • For IF/ICC: 1:50-1:500, validated in A549 cells and rat brain tissue

• Detection systems: For unconjugated antibodies, use appropriate secondary antibodies based on the host species (typically rabbit IgG for most commercial FOXO6 antibodies). For HRP-conjugated antibodies, optimize the substrate concentration and development time for optimal signal-to-noise ratio .

• Counterstaining: Nuclear counterstains are particularly useful as FOXO6 can show both nuclear and cytoplasmic localization depending on cellular context and activation state.

What methodological approaches are recommended for studying FOXO6 expression in different tissue types?

Different tissues require tissue-specific methodological considerations:

• Brain tissue: Given high FOXO6 expression in neural tissues, particularly during development , use mild fixation (4% PFA for 24h) and employ step-wise rehydration protocols to preserve tissue morphology. Confocal microscopy with z-stacking is recommended for detailed localization studies.

• Liver/Hepatocellular carcinoma: Due to FOXO6's association with HCC progression , implement dual staining with proliferation markers (like Ki-67) or glycolytic enzymes. For HCC tissues, comparison with adjacent non-tumor tissue is essential for accurate expression analysis.

• Cardiac tissue: When studying FOXO6 in cardiac pathology , co-staining with cardiomyocyte markers is recommended. Research has shown that FOXO6 overexpression in cardiomyocytes leads to cardiac dysfunction (decreased ejection fraction and fraction shortening), particularly under Ang-II treatment conditions .

• Craniofacial tissues: For developmental studies, staged tissue collection is critical as FOXO6 expression patterns change significantly during embryogenesis—from undetectable at E10.5 to specific expression in maxilla, mandible, incisors, molars, and palate by E18.5 in mouse models .

How can FOXO6 antibodies be utilized for studying protein-protein interactions and signaling pathways?

Advanced research into FOXO6 protein interactions and signaling requires specialized techniques:

• Co-immunoprecipitation (Co-IP): FOXO6 antibodies can be used for Co-IP to identify interaction partners. The recommended antibody amount is 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate . This approach has been successfully used to investigate FOXO6 interactions with components of the PI3K/Akt pathway .

• Chromatin Immunoprecipitation (ChIP): For studying FOXO6's role as a transcription factor, ChIP assays using FOXO6 antibodies can identify genomic binding sites. Published applications have successfully employed this technique using polyclonal FOXO6 antibodies .

• Signaling pathway analysis: To study FOXO6's role in the PI3K/Akt pathway:

  • Use pathway inhibitors (e.g., LY294002 for PI3K inhibition) or activators (e.g., 740Y-P for PI3K activation) in conjunction with FOXO6 knockdown or overexpression

  • Monitor changes in downstream targets using multiplexed Western blotting

  • Research indicates that FOXO6 knockdown inactivates PI3K and Akt proteins, thereby inhibiting the PI3K/Akt signaling pathway

What are the methodological challenges in studying FOXO6 phosphorylation states and how can they be addressed?

Studying FOXO6 phosphorylation presents several technical challenges:

• Phospho-specific antibodies: Use phospho-specific antibodies that target key regulatory sites, such as the FOXO6 (phospho S184) antibody . These antibodies require careful validation using phosphatase treatments as negative controls.

• Sample preparation: Critical steps include:

  • Rapid tissue/cell lysis in buffers containing phosphatase inhibitors

  • Maintaining samples at 4°C throughout processing

  • Using SDS-PAGE with Phos-tag™ acrylamide for improved separation of phosphorylated species

• Detection challenges: Phosphorylation can alter epitope accessibility, potentially affecting antibody binding. Using antibodies targeting different regions of FOXO6 can help ensure detection regardless of phosphorylation state.

• Functional correlation: Correlate phosphorylation data with functional outcomes by combining with techniques like gene reporter assays, as FOXO6 phosphorylation can affect its transcriptional activity.

How should researchers approach FOXO6 knockdown/overexpression studies in combination with antibody detection?

Comprehensive FOXO6 functional studies require careful experimental design:

• Knockdown validation: When performing FOXO6 knockdown:

  • Validate siRNA efficiency using both mRNA (RT-PCR) and protein (Western blot) measurements

  • Research has shown that designed FOXO6 siRNA sequences can effectively reduce FOXO6 expression in both cardiomyocytes and fibroblasts

  • For in vivo studies, consider conditional knockout systems as used in cardiac-specific FOXO6 deletion models

• Overexpression systems:

  • For cellular studies, adenoviral vectors have been successfully used to overexpress FOXO6 in cardiomyocytes

  • For animal models, cardiac-specific FOXO6-overexpressing myocardial tissues have been generated to study pathological remodeling

  • Validate overexpression using antibodies that can distinguish between endogenous and tagged proteins

• Methodological considerations for combined studies:

  • When studying the effects of FOXO6 manipulation on drug resistance (e.g., paclitaxel resistance in HCC), combine gene manipulation with drug treatment protocols

  • For analyzing glycolysis changes, pair FOXO6 manipulation with metabolic assays

  • In cardiac studies, combine FOXO6 manipulation with Ang-II treatment to investigate pathological remodeling

What are common issues when using HRP-conjugated FOXO6 antibodies and how can they be resolved?

Common issues and solutions when working with HRP-conjugated FOXO6 antibodies include:

• High background signal:

  • Increase blocking time (5% BSA or milk for 2 hours at room temperature)

  • Optimize antibody dilution (start with manufacturer's recommendation and test 2-3 additional dilutions)

  • Add 0.05-0.1% Tween-20 to washing buffers

  • Consider adding 0.05% Tween-20 to antibody diluent

• Weak or absent signal:

  • Check substrate freshness and reaction time

  • Verify protein transfer efficiency with reversible staining

  • Ensure proper storage of conjugated antibody (typically -20°C, avoid repeated freeze-thaw cycles)

  • Note that FOXO6 is observed at 70-80 kDa despite a calculated weight of 51 kDa

• Non-specific bands:

  • Increase stringency of washing steps (more washes, longer duration)

  • Pre-adsorb antibody with non-specific proteins

  • Consider using gradient gels to better separate proteins in the target size range

• Signal degradation during storage:

  • Store HRP-conjugated antibodies at -20°C or -80°C

  • Aliquot to avoid repeated freeze-thaw cycles

  • Add 50% glycerol to stabilize during storage

How can researchers optimize FOXO6 antibody protocols when working with different experimental models?

Optimization strategies for different model systems:

• Cell line models:

  • For human cell lines (HeLa, HepG2, A549): Standard lysis protocols with RIPA buffer are generally effective

  • For muscle cells (C2C12): More stringent lysis conditions may be needed due to higher protein content

  • Fixation for IF: 4% paraformaldehyde for 15 minutes is typically sufficient

• Tissue samples:

  • Mouse/rat brain tissue: Requires gentle homogenization and may benefit from specialized extraction buffers containing high salt (300-500mM NaCl) to extract nuclear proteins

  • Liver tissue: Contains high levels of proteases, so additional protease inhibitors are recommended

  • Cardiac tissue: Can be challenging due to high collagen content; mechanical disruption combined with enzymatic digestion may improve results

• Protocol optimization:

  • For IHC: Antigen retrieval is critical; TE buffer at pH 9.0 has shown superior results compared to citrate buffer for FOXO6 detection

  • For IF: Signal amplification systems may be beneficial, particularly in tissues with lower expression levels

What are the critical considerations for interpreting FOXO6 localization data from immunostaining experiments?

Accurate interpretation of FOXO6 localization requires understanding several critical factors:

• Nuclear vs. cytoplasmic distribution:

  • FOXO6 can shuttle between nucleus and cytoplasm depending on phosphorylation state

  • Proper controls for nuclear staining (DAPI) and cytoplasmic markers are essential

  • Fixation methods can artificially alter localization; compare multiple fixation protocols

• Cell type-specific patterns:

  • In neural tissues, FOXO6 shows predominantly nuclear localization

  • In cardiomyocytes, FOXO6 expression is significantly higher than in non-cardiomyocytes under both basal and Ang-II treatment conditions

  • Expression patterns change during development, with specific temporal and spatial regulation in craniofacial tissues

• Quantification approaches:

  • Use nuclear/cytoplasmic intensity ratios rather than binary classification

  • Employ Z-stack imaging to ensure complete visualization of subcellular compartments

  • Consider co-localization analysis with compartment-specific markers for quantitative assessment

How are FOXO6 antibodies being utilized to study its role in cancer metabolism and drug resistance?

FOXO6 antibodies are proving invaluable in elucidating its role in cancer:

• Hepatocellular carcinoma studies:

  • FOXO6 is highly expressed in HCC tissues and associated with patient prognosis

  • Antibody-based detection reveals that FOXO6 knockdown inhibits proliferation and invasion while inducing apoptosis in HCC cells

  • Western blot analysis using FOXO6 antibodies has demonstrated that FOXO6 knockdown suppresses glycolysis and reverses resistance to chemotherapy in Hep3B/PTX cells by inactivating the PI3K/Akt signaling pathway

• Methodological approaches:

  • Combining FOXO6 expression analysis (via antibodies) with metabolic assays to correlate expression with glycolytic activity

  • Using phospho-specific antibodies to track PI3K/Akt pathway activation in relation to FOXO6 levels

  • Employing FOXO6 antibodies in tissue microarrays to correlate expression with clinical outcomes and treatment response

• Emerging research directions:

  • Development of therapeutic strategies targeting FOXO6-regulated metabolic pathways

  • Investigation of FOXO6 as a biomarker for chemotherapy resistance, particularly to paclitaxel in HCC

What methodological advances are improving the study of FOXO6 in cardiac pathology and remodeling?

Recent methodological advances have enhanced our understanding of FOXO6 in cardiac pathology:

• Cell-specific expression profiling:

  • Western blotting indicates significantly greater expression of FOXO6 in cardiomyocytes than in non-cardiomyocytes

  • Immunofluorescence intensity of FOXO6 increases with time after Ang-II treatment in TAC-operated versus sham-operated wild-type mice

• Tissue-specific genetic manipulation:

  • Cardiomyocyte-specific FoxO6 knockout (FoxO6 cKO) and overexpression models

  • Expression analysis reveals that FoxO6 deficiency significantly reduces cardiac dysfunction markers after Ang-II treatment

  • Immunofluorescence staining of α-SMA-positive cells demonstrates that FoxO6 deficiency decreases fibroblast differentiation induced by Ang-II treatment

• Integrated pathway analysis:

  • Combination of FOXO6 antibody techniques with TGF-β1 measurement

  • RNA sequencing combined with ChIP-seq to identify FOXO6 targets in cardiac tissue

  • Discovery that FOXO6 regulates the Kif15/TGF-β1 axis in cardiac remodeling

How can researchers advance developmental studies of FOXO6 in craniofacial growth using antibody-based techniques?

Advanced developmental studies of FOXO6 in craniofacial growth require sophisticated approaches:

• Temporal-spatial expression mapping:

  • X-gal staining of staged embryos reveals that FOXO6 expression changes dramatically during development :

    • E10.5: Not detectably expressed

    • E12.5: Expressed in brain, somites, and craniofacial region

    • E14.5: Present in brain, somites, posterior maxilla and mandible

    • E18.5: Increased expression in craniofacial regions including maxilla, mandible, incisor, molar, and palate

• Integration with signaling pathways:

  • FOXO6 has been identified as an activator of Hippo signaling regulating neonatal growth of the face

  • Antibody-based co-immunoprecipitation to identify direct interaction partners in the Hippo pathway

  • ChIP-seq approaches to identify FOXO6 binding sites in genes related to craniofacial development

• 3D imaging techniques:

  • Whole-mount immunostaining followed by tissue clearing and light sheet microscopy

  • Time-lapse imaging of FOXO6 expression in craniofacial explant cultures

  • Correlation of FOXO6 expression patterns with morphometric analysis of craniofacial growth