FOXO6 Antibody, Biotin conjugated

What is FOXO6 and why is it a target for antibody-based detection?

FOXO6 (Forkhead Box O6) functions as a transcriptional activator in several critical cellular processes. As a member of the forkhead family of transcription factors, FOXO6 plays an essential role in integrating insulin signaling with gluconeogenesis in the liver and also participates in neuronal migration during cortical development . FOXO6 has gained research interest due to its protective role in aging processes, particularly through attenuating oxidative stress . Antibody-based detection allows researchers to study these functions by visualizing FOXO6 expression patterns, subcellular localization, and post-translational modifications.

What are the main experimental applications for FOXO6 antibodies?

FOXO6 antibodies, including biotin-conjugated versions, are validated for multiple experimental applications:

Note that biotin conjugation enhances detection sensitivity across these applications by allowing streptavidin-based amplification systems .

Which tissue and cell types show significant FOXO6 expression?

Based on validated antibody studies, FOXO6 expression has been detected in:

• Neural tissues: Mouse brain tissue shows robust expression, with specific FOXO6 activity observed during cortical development

• Cell lines: C2C12 cells (mouse myoblast), A549 cells (human lung)

• Other tissues: Mouse lung tissue

• Pathological samples: Human gliomas tissue

When designing experiments, these expression patterns should inform your sample selection and controls.

How can I validate the specificity of a biotin-conjugated FOXO6 antibody?

Methodological approach to antibody validation should include:

• Western blot analysis: Compare FOXO6 detection in known positive samples (e.g., brain tissue) versus negative controls. The expected molecular weight for FOXO6 is 51-61 kDa (calculated), though observed migration is often at 70-80 kDa .

• Knockout/knockdown validation: Test antibody reactivity in samples where FOXO6 has been knocked down using siRNA. Research has demonstrated that two different siRNAs targeting different parts of FOXO6 mRNA (siFoxO6-1 and siFoxO6-2) effectively reduced FOXO6 expression .

• Cross-reactivity assessment: Confirm that the antibody does not cross-react with other FOXO family members. This is particularly important as FOXO family proteins share sequence homology. Some FOXO6 antibodies have been specifically tested to ensure no cross-reactivity with other FOXOs .

• Phospho-specific validation: For phospho-specific FOXO6 antibodies, compare untreated samples with phosphatase-treated samples .

What is the optimal protocol for using biotin-conjugated FOXO6 antibodies in immunofluorescence studies?

For optimal immunofluorescence results with biotin-conjugated FOXO6 antibodies:

• Cell preparation:

  • Seed cells (e.g., HEK293T) at 1 × 10^4 cells per well in a 12-well plate

  • Incubate for 24 hours

  • Fix in 4% paraformaldehyde solution for 15 minutes at room temperature

  • Wash with PBS buffer

  • Block with 3% normal goat serum

• Antibody incubation:

  • Apply biotin-conjugated FOXO6 antibody at 1:200-1:800 dilution in blocking buffer

  • Incubate overnight at 4°C

  • Wash thoroughly with TBS

• Detection:

  • Incubate with streptavidin conjugated to a fluorophore (e.g., Alexa Fluor 488) at 1:200 dilution

  • Incubate for 3 hours at room temperature

  • Co-stain nuclei with Hoechst 33342 (1:1,000)

• Analysis: Visualize using confocal laser scanning microscopy, examining both nuclear and cytoplasmic distribution of FOXO6 .

What storage conditions are recommended for maintaining biotin-conjugated FOXO6 antibody activity?

To preserve biotin-conjugated FOXO6 antibody activity:

• Store at -20°C, with some manufacturers recommending -80°C for long-term storage

• Avoid repeated freeze-thaw cycles; aliquot upon receipt for single-use portions

• Store in the provided buffer, typically PBS with 50% glycerol and 0.02% sodium azide at pH 7.3-7.4

• Some preparations contain preservatives like ProClin 300 (0.03%), which should be handled with caution as a hazardous substance

• Return to proper storage temperature immediately after use

• Upon thawing, mix gently before use

How can biotin-conjugated FOXO6 antibodies be used for chromatin immunoprecipitation (ChIP) studies?

For ChIP studies investigating FOXO6 binding to target gene promoters:

• Sample preparation:

  • Culture cells with appropriate treatments (e.g., H₂O₂ with or without FOXO6 viruses)

  • Cross-link proteins to DNA with 1% formaldehyde

  • Sonicate samples using an ultrasonicator (e.g., VCX-600) at 30% maximum power, five times for 20 seconds

  • Centrifuge at 18,000 × g for 10 minutes

• Immunoprecipitation:

  • Incubate supernatant with 5-μg biotin-conjugated FOXO6 antibody

  • Perform immunoprecipitation using a ChIP assay kit (e.g., Upstate Biotechnology)

  • Analyze immunoprecipitates by PCR using promoter-specific primers

This method has been successfully used to demonstrate FOXO6 binding to specific DNA sequences, such as the catalase promoter (GenBank accession number AY545477) and Daf-16-binding elements (DBEs) in the Plxna4 promoter .

What is the significance of phosphorylated FOXO6 (pS184) detection and how does biotin conjugation enhance this analysis?

FOXO6 phosphorylation at S184 is a critical regulatory mechanism in response to insulin signaling:

• Biological significance:

  • Phosphorylation of FOXO6 at S184 occurs in response to insulin stimulation

  • This modification regulates FOXO6's role in hepatic gluconeogenesis

  • In mice, elevated FOXO6 activity increases gluconeogenesis, raising fasting blood glucose levels

• Detection advantages with biotin conjugation:

  • Higher sensitivity for detecting low-abundance phosphorylated forms

  • Ability to use streptavidin-based amplification techniques

  • Compatible with multiplex analysis when combined with other non-biotin antibodies

  • Reduced background when using phospho-specific antibodies generated against the S184 region (amino acids 182-195 of mouse FOXO6)

• Validation:

  • Phospho-specific antibodies show selective reactivity with phosphorylated FOXO6 in insulin-treated samples

  • Specificity can be confirmed by treating samples with alkaline phosphatase (AP)

How can we investigate FOXO6's role in oxidative stress response using biotin-conjugated antibodies?

To study FOXO6's role in oxidative stress response:

• Luciferase reporter assay setup:

  • Transfect cells (e.g., HEK293T) with catalase-Luc vector containing FoxO binding sequences

  • Use Lipofectamine 2000 with 1 μg DNA/0.5 μl Lipofectamine complexes

  • Treat with FoxO6 virus (100 MOI) or FoxO6-siRNA (100 MOI)

  • Induce oxidative stress with 100 μM H₂O₂ for 2 hours

  • Measure luciferase activity using a luminometer

• ChIP analysis of antioxidant gene promoters:

  • Use biotin-conjugated FOXO6 antibodies to immunoprecipitate FOXO6-bound chromatin

  • Analyze binding to promoters of antioxidant enzymes like catalase

  • Compare binding under normal and oxidative stress conditions

• Western blot analysis:

  • Detect both total and phosphorylated FOXO6 in response to oxidative stress

  • Examine correlations between FOXO6 phosphorylation state and antioxidant enzyme expression

  • Research has shown that FOXO6 has a protective role in aging through its ability to attenuate oxidative stress

What are common issues with biotin-conjugated antibodies in multi-step detection protocols?

When working with biotin-conjugated FOXO6 antibodies, researchers may encounter:

• High background signal:

  • Cause: Endogenous biotin in samples, particularly in biotin-rich tissues like liver and kidney

  • Solution: Include an avidin/biotin blocking step before primary antibody incubation

  • Method: Incubate with avidin solution for 15 minutes, wash, then incubate with biotin solution for 15 minutes

• Cross-reactivity with other FOXO family members:

  • Cause: Sequence homology between FOXO proteins

  • Solution: Validate antibody specificity with both positive and negative controls

  • Data shows specific antibodies (e.g., anti-phospho-FOXO6) do not cross-react with other FOXOs

• Inconsistent signal amplification:

  • Cause: Variable streptavidin-biotin binding

  • Solution: Optimize streptavidin-HRP or streptavidin-fluorophore concentration

  • Recommended: Titrate detection reagents for each experimental system

How can I determine the optimal working dilution for biotin-conjugated FOXO6 antibodies across different applications?

A systematic approach to antibody titration:

• Initial range finding:

  • For Western blotting: Test dilutions from 1:500 to 1:2000

  • For IHC: Test dilutions from 1:100 to 1:1000

  • For IF/ICC: Test dilutions from 1:100 to 1:800

• Optimizing signal-to-noise ratio:

  • Prepare a titration series using 2-fold dilutions

  • Include positive control samples with known FOXO6 expression (e.g., brain tissue)

  • Include negative controls (secondary antibody only)

  • Evaluate both signal intensity and background levels

• Application-specific considerations:

  • For Western blot: Consider loading controls and protein amount (typically 10-20 μg)

  • For IHC: Evaluate antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • For ChIP: Optimize antibody-to-chromatin ratio (typically 5 μg antibody per IP)

• Validation: Once optimal dilution is determined, validate with biological replicates to ensure reproducibility.

What considerations are important when using biotin-conjugated FOXO6 antibodies in tissues with high endogenous biotin?

When working with biotin-rich tissues:

• Pre-blocking strategy:

  • Implement an avidin-biotin blocking step:

    • Incubate sections with avidin solution (15 minutes)

    • Wash thoroughly

    • Incubate with biotin solution (15 minutes)

    • Proceed with normal staining protocol

• Alternative detection systems:

  • Consider tyramide signal amplification (TSA) systems that are less affected by endogenous biotin

  • Use alternative secondary detection methods if biotin interference persists

• Tissue-specific controls:

  • Always include tissue-matched negative controls processed without primary antibody

  • Consider using FOXO6 knockout or knockdown tissues as additional controls

• Signal verification:

  • Confirm FOXO6 signal pattern with an unconjugated FOXO6 antibody detected through non-biotin methods

  • Compare patterns to published expression data for FOXO6 in tissues of interest

How can biotin-conjugated FOXO6 antibodies be used to study neurodevelopmental disorders?

FOXO6 plays crucial roles in neuronal migration and cortical development, making it relevant to neurodevelopmental disorders:

• Cortical migration studies:

  • FOXO6 knockdown via siRNA results in hampered radial migration in developing cortex

  • At E18 (embryonic day 18), FOXO6 siRNA electroporated cortices showed 48% of transfected cells in the intermediate zone compared to 27% in controls

  • Only 35.2% of cells reached the cortical plate in FOXO6-knockdown versus 66.9% in controls

• Experimental approach:

  • Use biotin-conjugated FOXO6 antibodies to track expression in brain development

  • Perform IF/IHC studies comparing normal and disease model tissues

  • Employ ChIP analysis to identify FOXO6 target genes in neural tissues

• Plxna4 pathway investigation:

  • FOXO6 regulates Plxna4 expression during cortical development

  • ChIP analysis using biotin-conjugated FOXO6 antibodies can confirm binding to Daf-16-binding elements (DBEs) in the Plxna4 promoter

  • This pathway is critical for proper neuronal migration

What is the significance of FOXO6 in metabolic disorders and how can biotin-conjugated antibodies advance this research?

FOXO6 integrates insulin signaling with gluconeogenesis, positioning it as a key factor in metabolic disorders:

• Metabolic regulation:

  • Elevated FOXO6 activity in the liver augments gluconeogenesis, raising fasting blood glucose levels

  • Insulin stimulation triggers FOXO6 phosphorylation at S184

• Research applications:

  • Use biotin-conjugated antibodies to track FOXO6 expression and phosphorylation status in metabolic tissues

  • Perform ChIP studies to identify gluconeogenic gene targets

  • Compare FOXO6 binding patterns in normal versus insulin-resistant states

• Phosphorylation detection:

  • Employ phospho-specific antibodies (pS184) to monitor insulin responsiveness

  • Biotin conjugation enhances detection sensitivity for monitoring phosphorylation dynamics

  • Compare phosphorylation patterns using Western blot analysis of tissues from normal and metabolic disorder models

How can biotin-conjugated FOXO6 antibodies be utilized in aging and longevity studies?

FOXO6 has demonstrated protective roles in aging processes, particularly through oxidative stress regulation:

• Aging biomarker analysis:

  • Track FOXO6 expression and phosphorylation changes during aging

  • Compare FOXO6 activity in normal aging versus calorie restriction models

  • Research shows FOXO6's protective role in aging may relate to its ability to attenuate oxidative stress

• Catalase regulation investigation:

  • Use ChIP with biotin-conjugated FOXO6 antibodies to study binding to the catalase promoter

  • Analyze age-dependent changes in FOXO6-mediated antioxidant responses

  • Correlate findings with oxidative damage markers

• Caloric restriction models:

  • Compare FOXO6 phosphorylation and activity in ad libitum versus calorie-restricted animals

  • Investigate FOXO6 target gene expression profiles under different dietary conditions

What are the latest technical advances in using biotin-conjugated antibodies for multiplexed FOXO family detection?

Recent developments in multiplexed detection systems:

• Spectrally distinct fluorophores:

  • Combine biotin-conjugated FOXO6 antibodies with directly labeled antibodies against other FOXO family members

  • Use streptavidin conjugated to spectrally distinct fluorophores (e.g., Alexa Fluor 488, 555, 647)

  • This allows simultaneous visualization of multiple FOXO proteins in the same sample

• Sequential detection protocols:

  • Apply biotin-based tyramide signal amplification for low-abundance targets

  • Combine with conventional detection methods for more abundant targets

  • This approach maximizes sensitivity while maintaining specificity

• Spatial profiling applications:

  • Biotin-conjugated FOXO6 antibodies can be integrated into spatial transcriptomics/proteomics platforms

  • This enables correlation of FOXO6 protein localization with gene expression patterns

These methodological approaches provide researchers with powerful tools to study the complex regulatory networks involving FOXO6 in various physiological and pathological contexts.