Phospho-FOXO4 (S262) Antibody

What is FOXO4 and what role does phosphorylation at S262 play in its function?

FOXO4 (Forkhead box protein O4, also known as AFX or AFX1) is a transcription factor involved in regulating the insulin signaling pathway. It binds to insulin-response elements (IREs) and activates transcription of IGFBP1. FOXO4 down-regulates expression of HIF1A and suppresses hypoxia-induced transcriptional activation of HIF1A-modulated genes . It also plays roles in negative regulation of the cell cycle and increased proteasome activity in embryonic stem cells (ESCs) by activating expression of PSMD11, leading to enhanced assembly of the 26S proteasome .

Phosphorylation at S262 is a key regulatory mechanism that affects FOXO4's activity and cellular localization. When FOXO4 is phosphorylated at S262, it typically translocates from the nucleus to the cytoplasm, effectively inhibiting its transcriptional activity . This phosphorylation site is therefore crucial for controlling FOXO4's ability to regulate its target genes in response to cellular signals, particularly insulin pathway activation.

How does the phosphorylation state of FOXO4 at S262 affect its cellular localization?

The phosphorylation state of FOXO4 at S262 directly influences its cellular compartmentalization. According to established research, when FOXO4 is phosphorylated, it translocates from the nucleus to the cytoplasm . Conversely, dephosphorylation triggers nuclear translocation, allowing FOXO4 to access and regulate its target genes.

This nucleocytoplasmic shuttling represents a rapid mechanism to control FOXO4's transcriptional activities. When insulin or growth factor signaling is active, FOXO4 becomes phosphorylated at S262 (among other sites), causing its export from the nucleus and preventing it from activating its target genes . This phosphorylation-dependent localization is part of a complex regulatory network, as other modifications also influence FOXO4 localization - monoubiquitination increases nuclear localization, while deubiquitination promotes translocation from the nucleus to the cytoplasm .

What tissues express FOXO4, and how does this influence experimental design when using Phospho-FOXO4 (S262) antibodies?

FOXO4 is expressed across multiple human tissues, with presence detected in heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas . Notably, isoform zeta of FOXO4 is most abundant in the liver, kidney, and pancreas . This tissue distribution has several important implications for experimental design:

• Control tissue selection: Researchers should select appropriate positive control tissues when validating Phospho-FOXO4 (S262) antibodies. Liver, kidney, or pancreas samples would be ideal positive controls due to their higher expression levels .

• Tissue-specific isoform considerations: Since isoform zeta is predominant in specific tissues, researchers should account for potential isoform-specific effects when interpreting results across different tissue types .

• Background signal optimization: Different tissues express varying levels of FOXO4, requiring optimization of antibody dilutions for each tissue type. The recommended dilutions range from 1:500-1:2000 for Western blotting according to commercial antibodies .

• Knockdown validation controls: When designing knockdown or knockout experiments to validate antibody specificity, researchers should choose cell lines derived from tissues with notable FOXO4 expression to ensure adequate starting signal.

What are the common applications for Phospho-FOXO4 (S262) antibodies in research?

Phospho-FOXO4 (S262) antibodies are utilized in several key research applications:

• Western Blotting (WB): This is the most validated application, with antibodies typically used at dilutions between 1:500-1:10000 depending on the specific product . Western blotting allows researchers to quantify relative levels of phosphorylated FOXO4 under different experimental conditions.

• Immunohistochemistry (IHC): Some antibodies are suitable for detecting phosphorylated FOXO4 in tissue sections, enabling visualization of its distribution in different cell types and tissues . This application typically requires specific sample preparation, such as heat-mediated antigen retrieval with sodium citrate buffer (pH 6.0) .

• Insulin Signaling Research: Given FOXO4's role in insulin signaling, phospho-specific antibodies are valuable tools for studying how insulin and related pathways regulate FOXO4 activity through phosphorylation at S262 .

• Cell Cycle Regulation Studies: Since FOXO4 is involved in negative regulation of the cell cycle, phospho-specific antibodies help examine how its phosphorylation state changes during different cell cycle phases .

• Cancer Research: FOXO4 is involved in chromosomal aberrations in acute leukemias, and phospho-specific antibodies can help investigate its role in oncogenic pathways .

What is the relationship between FOXO4 phosphorylation and the insulin signaling pathway?

FOXO4 is a key downstream target in the insulin signaling pathway. The relationship between FOXO4 phosphorylation at S262 and insulin signaling involves several interconnected processes:

• Insulin Receptor Activation: Insulin binding to its receptor initiates a signaling cascade that ultimately leads to FOXO4 phosphorylation, including at the S262 site .

• Transcriptional Regulation: When active (unphosphorylated) in the nucleus, FOXO4 binds to insulin-response elements (IREs) and can activate transcription of genes like IGFBP1 (Insulin-like Growth Factor Binding Protein 1) .

• Nuclear Exclusion: Phosphorylation of FOXO4 at S262 promotes its translocation from the nucleus to the cytoplasm, effectively inactivating its transcriptional activity . This represents a mechanism by which insulin signaling inhibits FOXO4-mediated gene expression.

• Metabolic Control: Through this phosphorylation-mediated regulation, insulin controls FOXO4-dependent transcriptional programs involved in metabolism, cell growth, and survival .

Researchers use Phospho-FOXO4 (S262) antibodies to monitor how manipulations of the insulin signaling pathway affect FOXO4 phosphorylation status, helping to elucidate the molecular mechanisms connecting insulin signaling to downstream cellular responses.

How can Phospho-FOXO4 (S262) antibodies be used to study the relationship between FOXO4 and HIF1A-mediated responses to hypoxia?

The research literature indicates that FOXO4 down-regulates expression of HIF1A and suppresses hypoxia-induced transcriptional activation of HIF1A-modulated genes . Phospho-FOXO4 (S262) antibodies can be strategically deployed to investigate this relationship through several methodological approaches:

• Comparative Phosphorylation Analysis: Researchers can use these antibodies to monitor changes in FOXO4 S262 phosphorylation status under normoxic versus hypoxic conditions, revealing how oxygen tension affects FOXO4 regulation.

• Phosphorylation-Dependent HIF1A Regulation: By combining Phospho-FOXO4 (S262) antibody detection with HIF1A expression analysis, researchers can determine whether the phosphorylation state of FOXO4 correlates with its ability to suppress HIF1A.

• ChIP-Seq Applications: Chromatin immunoprecipitation followed by sequencing using Phospho-FOXO4 (S262) antibodies can identify genomic binding sites of phosphorylated FOXO4 during hypoxia, potentially revealing direct targets related to hypoxia response.

• Phosphomimetic/Phosphodeficient Mutant Studies: Researchers can generate FOXO4 mutants that either mimic (S262D/E) or prevent (S262A) phosphorylation at S262, then use Phospho-FOXO4 (S262) antibodies to confirm the mutants' effects and examine their impact on HIF1A-mediated transcription.

• Pharmacological Manipulation: Treatment with kinase inhibitors or phosphatase activators can alter FOXO4 phosphorylation, and Phospho-FOXO4 (S262) antibodies can monitor these changes and correlate them with hypoxia responses.

This methodological framework helps dissect the complex relationship between FOXO4 phosphorylation at S262 and hypoxia signaling pathways, potentially revealing new therapeutic targets for hypoxia-related conditions.

What experimental controls are crucial when using Phospho-FOXO4 (S262) antibodies to ensure specificity of phosphorylation state detection?

When using Phospho-FOXO4 (S262) antibodies, researchers should implement several critical experimental controls to ensure reliable and specific detection:

• Phosphatase Treatment Control: Treating samples with alkaline phosphatase removes phosphorylation and should eliminate signal from a truly phospho-specific antibody. This negative control confirms phospho-specificity, as demonstrated in published protocols showing signal loss after alkaline phosphatase treatment .

• Non-Transfected Cell Control: Including lysates from cells that don't express FOXO4 helps establish background signal levels. This approach is evident in commercial antibody validation where non-transfected 293T cells are used as controls .

• Phosphomimetic and Phosphodeficient Mutants: Using FOXO4 constructs with S262A (cannot be phosphorylated) or S262D/E (mimics phosphorylation) mutations can validate antibody specificity by showing expected reactivity patterns.

• Competing Phosphopeptide: Pre-incubating the antibody with the phosphopeptide used as immunogen should block specific binding and eliminate signal. Several of the antibodies in the search results use synthetic phosphopeptides as immunogens .

• siRNA/CRISPR Knockdown: Reducing FOXO4 expression through genetic approaches should proportionally reduce signal if the antibody is specific.

• Stimulation Controls: Using known activators or inhibitors of pathways that regulate FOXO4 phosphorylation (like insulin pathway modulators) should produce predictable changes in signal.

• Multiple Antibody Validation: Using different antibodies targeting the same phospho-site from different vendors can increase confidence in results. The search results show multiple commercially available antibodies that could be used for this purpose .

Implementation of these controls ensures that observed signals genuinely represent S262-phosphorylated FOXO4, rather than non-specific binding or cross-reactivity with other proteins or phosphorylation sites.

How might Phospho-FOXO4 (S262) antibodies be employed to investigate the role of FOXO4 in proteasome assembly and activity in embryonic stem cells?

FOXO4 is involved in increased proteasome activity in embryonic stem cells (ESCs) by activating expression of PSMD11, leading to enhanced assembly of the 26S proteasome and higher proteasome activity . Phospho-FOXO4 (S262) antibodies can be strategically employed to investigate this role through several approaches:

• Phosphorylation Status During Differentiation: Using Phospho-FOXO4 (S262) antibodies to track changes in FOXO4 phosphorylation during ESC differentiation can reveal correlations between phosphorylation state and proteasome activity changes.

• ChIP-Seq Analysis: Chromatin immunoprecipitation with Phospho-FOXO4 (S262) antibodies followed by sequencing can identify genomic binding sites of phosphorylated FOXO4 in ESCs, potentially revealing direct regulation of PSMD11 or other proteasome-related genes.

• Phosphorylation-Dependent Transcriptional Activity: By combining Phospho-FOXO4 (S262) detection with PSMD11 expression analysis, researchers can determine whether FOXO4's phosphorylation state correlates with its ability to drive PSMD11 transcription.

• Subcellular Fractionation: Using these antibodies in conjunction with subcellular fractionation can reveal whether phosphorylated FOXO4 associates with proteasome components in specific cellular compartments. This is particularly relevant given FOXO4's known translocation between nucleus and cytoplasm depending on phosphorylation status .

• Proteasome Activity Correlation: Researchers can manipulate signaling pathways that affect FOXO4 S262 phosphorylation, then use the antibodies to confirm phosphorylation changes and correlate these with measured proteasome activity.

These approaches provide a comprehensive framework for dissecting the specific role of phosphorylated FOXO4 at S262 in regulating proteasome function in ESCs, with potential implications for understanding stem cell biology and pluripotency maintenance.

What are the technical challenges in differentiating between phosphorylated FOXO4 at S262 and other phosphorylation sites or related FOXO family members?

Several technical challenges exist when attempting to differentiate between FOXO4 phosphorylated at S262 and other phosphorylation sites or related FOXO family members:

• Sequence Homology: The FOXO family (FOXO1, FOXO3, FOXO4, and FOXO6) shares significant sequence homology, particularly in functional domains. This can lead to cross-reactivity of antibodies designed to target specific phosphorylation sites. For example, search result describes a dual-specificity antibody that recognizes both "Phospho-FoxO1 (Ser319)/FoxO4 (Ser262)," highlighting this challenge.

• Multiple Phosphorylation Sites: FOXO4 contains multiple phosphorylation sites beyond S262, some in similar sequence contexts, challenging antibody specificity. The precise immunogen sequence is often proprietary, as noted in search result .

• Epitope Masking: Protein-protein interactions or conformational changes may mask the S262 phosphorylation site, reducing antibody accessibility and producing false negatives.

• Antibody Validation Limitations: As seen in search result , antibodies are often validated using overexpression systems rather than endogenous protein. The search results show validation using transfected 293T cells, which may not fully represent physiological conditions.

• Detection Method Sensitivity: Western blotting may not have sufficient sensitivity to detect low levels of endogenous phosphorylated FOXO4, as suggested by the "Transfected Only" sensitivity noted in search result .

• Simultaneous Modifications: Co-occurrence of phosphorylation at S262 with other post-translational modifications (like ubiquitination mentioned in search result ) may alter epitope recognition.

To address these challenges, researchers should:

• Use multiple antibodies targeting different epitopes

• Implement the comprehensive controls described in section 2.2

• Consider using mass spectrometry-based approaches for unambiguous identification

• Combine genetic approaches (site-directed mutagenesis) with antibody-based detection

How can researchers effectively use Phospho-FOXO4 (S262) antibodies to study the implications of FOXO4 chromosomal aberrations in acute leukemias?

Chromosomal aberrations involving FOXO4, particularly translocation t(X;11)(q13;q23) with MLL/HRX, are found in acute leukemias . Researchers can effectively use Phospho-FOXO4 (S262) antibodies to study the implications of these aberrations through several methodological approaches:

• Comparative Phosphorylation Analysis: Researchers can compare S262 phosphorylation patterns between leukemia cells carrying FOXO4 translocations and normal counterparts, potentially revealing altered regulation of this phosphorylation site in leukemia.

• Fusion Protein Phosphorylation: For MLL-FOXO4 fusion proteins, Phospho-FOXO4 (S262) antibodies can determine whether the fusion retains this phosphorylation site and how its phosphorylation status affects function. The search results indicate that a chromosomal aberration involving FOXO4 is found in acute leukemias with translocation t(X;11)(q13;q23) with MLL/HRX .

• Signaling Pathway Assessment: Using these antibodies to monitor FOXO4 phosphorylation in response to various signaling inputs can reveal whether FOXO4 aberrations alter normal regulatory pathways in leukemic cells.

• Therapeutic Response Monitoring: Researchers can examine how treatments affecting FOXO4-related pathways influence S262 phosphorylation in leukemia cells with FOXO4 aberrations, potentially identifying biomarkers of response.

• Subcellular Localization Studies: Using these antibodies for immunofluorescence can determine whether FOXO4 translocations affect the normal phosphorylation-dependent shuttling between nucleus and cytoplasm described in search result .

• Target Gene Expression Correlation: Combining Phospho-FOXO4 (S262) detection with analysis of FOXO4 target genes can reveal whether aberrant FOXO4 retains normal transcriptional regulatory functions or gains new functions in the context of leukemia.

This comprehensive approach can provide insights into how FOXO4 chromosomal aberrations contribute to leukemogenesis and potentially identify novel therapeutic targets.

What sample preparation techniques optimize the detection of Phospho-FOXO4 (S262) in Western blotting experiments?

Optimizing the detection of Phospho-FOXO4 (S262) in Western blotting requires careful attention to sample preparation techniques. Based on the search results and standard phosphoprotein analysis methods, the following protocol elements are recommended:

• Phosphatase Inhibitors: Include a comprehensive phosphatase inhibitor cocktail in all buffers to preserve phosphorylation states. This is crucial since phosphorylation at S262 is sensitive to phosphatase activity as demonstrated in the control experiments in search result .

• Lysis Buffer Composition: Use a buffer containing detergents like NP-40 or Triton X-100 with protease inhibitors. Search result indicates the use of a buffer containing potassium phosphate, sodium chloride, glycerol, and sodium azide.

• Sample Denaturation: Heat samples at 95-100°C for 5 minutes in Laemmli buffer with SDS and reducing agents to ensure complete protein denaturation, which is important for antibody access to the phospho-epitope.

• Protein Concentration Determination: Load equal amounts of protein (typically 10-30 μg) per lane. Search result mentions using 10 μg of cell lysate per lane for optimal detection.

• Gel Percentage Optimization: Use 8-10% polyacrylamide gels for optimal resolution of FOXO4, which has a molecular weight of approximately 53-65 kDa as indicated in search results .

• Transfer Conditions: Use wet transfer methods with methanol-containing buffer for better transfer of phosphoproteins.

• Blocking Agent Selection: Use 5% BSA in TBST rather than milk, as milk contains phosphoproteins and phosphatases that can interfere with detection.

• Primary Antibody Dilution and Incubation: Based on search results, use dilutions between 1:500-1:10000 depending on the specific antibody . For example, search result recommends a 1:10000 dilution. Incubate overnight at 4°C for optimal results.

• Positive Control Inclusion: Include lysates from cells with known FOXO4 phosphorylation status, such as FOXO4-transfected 293T cells as mentioned in search result .

• Phosphatase-Treated Control: Include a sample treated with alkaline phosphatase as a negative control, as demonstrated in search result .

Following these specialized sample preparation techniques will help preserve the phosphorylation state of FOXO4 at S262 and optimize its detection in Western blotting experiments.

What factors influence the choice between monoclonal and polyclonal Phospho-FOXO4 (S262) antibodies for specific research applications?

The search results mention both monoclonal (search result ) and polyclonal (search result ) Phospho-FOXO4 (S262) antibodies. The choice between these types involves several considerations for specific research applications:

Factors Favoring Monoclonal Antibodies:

• Consistency Across Experiments:

  • Monoclonal antibodies provide higher batch-to-batch reproducibility

  • Search result describes the monoclonal antibody YP-mAb-01363 with defined properties including a 1 mg/ml concentration and ≥90% purity

• Higher Specificity for Phospho-Epitope:

  • Monoclonal antibodies target a single epitope, potentially offering greater discrimination between phosphorylated and non-phosphorylated forms

  • Search result states that the monoclonal antibody "detects endogenous levels of FoxO4 protein only when phosphorylated at S262"

• Reduced Background in Certain Applications:

  • Their high specificity can result in cleaner signals in applications like immunohistochemistry

  • This is valuable for tissue analyses like those mentioned in search result

• Quantitative Applications:

  • Better suited for quantitative assays where precise epitope recognition is essential

Factors Favoring Polyclonal Antibodies:

• Epitope Accessibility:

  • Polyclonal antibodies recognize multiple epitopes, offering better detection when conformational changes or protein interactions might mask some epitopes

  • This could be important for FOXO4, given its complex regulation involving nuclear-cytoplasmic shuttling

• Signal Amplification:

  • Multiple antibodies binding to different epitopes around the phospho-site can provide stronger signals for low-abundance proteins

  • This may be valuable since search result notes "SENSITIVITY: Transfected Only" for a FOXO4 phospho-antibody

• Tolerance to Fixation-Induced Epitope Alterations:

  • In applications like immunohistochemistry (shown in search result ), polyclonal antibodies may maintain reactivity even if some epitopes are altered by fixation

• Cross-Species Reactivity:

  • May offer better cross-species reactivity due to recognition of multiple epitopes

  • Search results mention antibodies with reactivity to human and mouse , which is valuable for comparative studies

Application-Specific Recommendations:

Researchers should consider these factors when selecting between monoclonal and polyclonal Phospho-FOXO4 (S262) antibodies for their specific research applications.

How can researchers quantitatively assess changes in FOXO4 phosphorylation at S262 in response to experimental manipulations?

Quantitatively assessing changes in FOXO4 phosphorylation at S262 requires rigorous methodology. Based on the search results and standard phosphoprotein analysis approaches, researchers should consider the following methods:

• Western Blot Quantification:

  • Perform Western blot using Phospho-FOXO4 (S262) antibodies at optimized dilutions (1:1000 to 1:10000 as mentioned in search results and )

  • Include total FOXO4 detection on the same samples (via stripping and reprobing or parallel blots)

  • Calculate phospho-FOXO4/total FOXO4 ratios using densitometry software

  • Use appropriate normalization controls

  • Search result provides an example of Western blot detection showing a band at approximately 9 kDa (although the predicted size is 53 kDa)

• Phospho-Flow Cytometry:

  • Use fluorescently-labeled Phospho-FOXO4 (S262) antibodies for flow cytometry

  • Simultaneously measure total FOXO4 with differently-labeled antibodies

  • This allows single-cell resolution of phosphorylation changes and detection of cell subpopulations

• Mass Spectrometry-Based Approaches:

  • Use stable isotope labeling coupled with phosphopeptide enrichment

  • Identify and quantify the specific phosphopeptide containing S262

  • This provides absolute quantification and can simultaneously measure multiple phosphorylation sites

• Quantitative Immunofluorescence:

  • Perform immunofluorescence staining with Phospho-FOXO4 (S262) antibodies

  • Co-stain for total FOXO4 with differently-labeled antibodies

  • Quantify phospho/total ratios at subcellular levels using confocal microscopy and image analysis

  • This provides spatial information about phosphorylation changes, which is particularly valuable given FOXO4's phosphorylation-dependent nuclear-cytoplasmic shuttling

• In Vitro Kinase Assays:

  • Develop in vitro kinase assays with purified kinases and FOXO4 substrates

  • Quantify S262 phosphorylation using Phospho-FOXO4 (S262) antibodies

  • This helps identify direct kinases responsible for S262 phosphorylation

For all these methods, researchers should include appropriate controls as discussed in section 3.2 and calculate statistical significance across multiple experiments to ensure reliable quantification of phosphorylation changes.

What strategies can be employed to overcome the limited sensitivity of Phospho-FOXO4 (S262) antibodies for detecting endogenous protein?

Several search results indicate that sensitivity may be a limitation when detecting endogenous levels of phosphorylated FOXO4 at S262. For instance, search result explicitly states "SENSITIVITY: Transfected Only." To overcome this limitation, researchers can employ several strategies:

• Signal Amplification Techniques:

  • Use enhanced chemiluminescence (ECL) substrates with higher sensitivity for Western blotting

  • Employ tyramide signal amplification (TSA) for immunohistochemistry or immunofluorescence

  • These methods can amplify weak signals from endogenous phosphorylated FOXO4

• Protein Enrichment Prior to Detection:

  • Perform immunoprecipitation with total FOXO4 antibodies before Western blotting with Phospho-FOXO4 (S262) antibodies

  • Use phosphoprotein enrichment techniques like IMAC (Immobilized Metal Affinity Chromatography) or TiO₂ (Titanium Dioxide) beads

  • These approaches concentrate the target protein, improving detection of low-abundance phosphorylated forms

• Optimal Cell/Tissue Selection:

  • Choose tissues with higher FOXO4 expression as indicated in search result (liver, kidney, pancreas)

  • Use cell types known to have active insulin signaling, which regulates FOXO4 phosphorylation

  • Proper tissue selection increases baseline levels of the target protein

• Stimulation to Increase Phosphorylation:

  • Treat cells with insulin or growth factors to increase S262 phosphorylation

  • Inhibit relevant phosphatases to preserve phosphorylation

  • These manipulations can increase the proportion of phosphorylated FOXO4, improving detection

• Alternative Detection Methods:

  • Consider using Phos-tag™ SDS-PAGE, which can separate phosphorylated from non-phosphorylated proteins without relying solely on antibody specificity

  • Employ mass spectrometry-based approaches, which can often detect phosphopeptides with higher sensitivity than antibody-based methods

  • These methods provide complementary approaches when antibody sensitivity is limiting

• Improved Sample Preparation:

  • Use rapid protein extraction methods with immediate addition of phosphatase inhibitors

  • Avoid freeze-thaw cycles that may affect phosphoprotein stability

  • These precautions help preserve the phosphorylated state of endogenous FOXO4

• Newer Generation Antibodies:

  • Monitor literature and antibody catalogs for newer Phospho-FOXO4 (S262) antibodies with improved sensitivity

  • Consider recombinant antibodies, which often offer better sensitivity and specificity, such as the recombinant monoclonal antibody mentioned in search results

Implementation of these strategies can substantially improve the detection of endogenous phosphorylated FOXO4 at S262, enabling more physiologically relevant research.

How can researchers determine the specificity of Phospho-FOXO4 (S262) antibodies across different species for comparative studies?

Determining the cross-species specificity of Phospho-FOXO4 (S262) antibodies is crucial for comparative studies across experimental models. The search results indicate variability in species reactivity, with some antibodies recognizing human and mouse FOXO4 . Researchers can employ several methodological approaches to evaluate and confirm cross-species specificity:

• Sequence Alignment Analysis:

  • Compare the amino acid sequence surrounding S262 across species

  • Higher conservation indicates greater likelihood of antibody cross-reactivity

  • For example, researchers should examine whether the phosphorylation-site motif is conserved between human, mouse, and other model organisms

• Positive and Negative Controls from Multiple Species:

  • Test the antibody on samples from multiple species with known FOXO4 expression

  • Include both wild-type samples and FOXO4 knockout tissues/cells from each species

  • Search result indicates human and mouse reactivity for the described antibody

• Phosphatase Treatment Validation Across Species:

  • Perform parallel phosphatase treatment controls on samples from different species

  • Verify that signal disappears in all species following phosphatase treatment

  • This confirms that the antibody is detecting phosphorylated epitopes in each species

• Recombinant Protein Standards:

  • Use purified recombinant phosphorylated FOXO4 proteins from different species as standards

  • Compare antibody reactivity across these standards under identical conditions

  • This provides direct quantitative comparison of antibody affinity across species

• Immunoprecipitation-Mass Spectrometry Validation:

  • Perform immunoprecipitation with the Phospho-FOXO4 (S262) antibody on samples from different species

  • Confirm the identity of precipitated proteins by mass spectrometry

  • Verify that the phosphorylation site is correctly identified in each species

• Overexpression Studies with Species-Specific Constructs:

  • Overexpress FOXO4 from different species in a cell line

  • Compare antibody detection efficiency across these constructs

  • This approach is particularly useful given that some antibodies are validated using overexpression systems, as seen in search result

• Literature and Vendor Validation Data Review:

  • Examine published studies that have used the antibody across species

  • Review vendor validation data for species reactivity claims

  • The search results provide information on validated species reactivity for commercial antibodies

• Western Blot Band Pattern Analysis:

  • Compare the molecular weight and band pattern of detected proteins across species

  • Confirm that these match the expected species-specific characteristics of FOXO4

  • Search results indicate expected molecular weights between 53-65 kDa, with some variation

By implementing these methodological approaches, researchers can confidently establish the cross-species specificity of Phospho-FOXO4 (S262) antibodies, ensuring reliable comparative studies across experimental models.