Phospho-RUNX1 (S435) Antibody

What is Phospho-RUNX1 (S435) Antibody and what does it detect?

Phospho-RUNX1 (S435) Antibody is a polyclonal antibody that specifically recognizes RUNX1 protein only when phosphorylated at serine residue 435. This antibody is raised in rabbits using synthesized phospho-peptides derived from human RUNX1 protein around the S435 phosphorylation site . The antibody detects endogenous levels of RUNX1 protein exclusively when this specific post-translational modification is present, making it an essential tool for studying RUNX1 phosphorylation state and related signaling pathways . RUNX1, also known as AML1 or CBFA2, is a transcription factor that plays critical roles in hematopoietic development and is frequently involved in leukemia-associated chromosomal translocations .

What applications is Phospho-RUNX1 (S435) Antibody validated for?

The Phospho-RUNX1 (S435) Antibody has been specifically validated for Western Blot (WB) and ELISA applications . Western blotting allows researchers to detect the phosphorylated protein in cell or tissue lysates, providing information about relative expression levels and phosphorylation status under different experimental conditions. ELISA applications enable quantitative detection of phosphorylated RUNX1 in prepared samples. While these are the validated applications, researchers should note that the antibody may work in other applications but would require additional validation by the end user before implementation in experimental protocols .

What are the optimal storage conditions for preserving antibody activity?

For optimal preservation of antibody activity, Phospho-RUNX1 (S435) Antibody should be stored at -20°C or -80°C immediately upon receipt . The antibody is supplied in a liquid formulation containing PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide as stabilizers . The high glycerol content prevents freezing at -20°C and helps maintain antibody stability. Researchers should avoid repeated freeze-thaw cycles as these can lead to protein denaturation and loss of antibody activity . For laboratories that frequently use the antibody, it is recommended to prepare working aliquots before freezing to minimize freeze-thaw cycles. The antibody remains stable for up to one year from the date of receipt when stored properly .

What dilution ranges are recommended for different applications?

The manufacturer-recommended dilution ranges for Phospho-RUNX1 (S435) Antibody vary by application:

These dilution recommendations serve as starting points and may require optimization based on specific experimental conditions, sample types, and detection systems employed . Researchers should perform a dilution series during initial experiments to determine the optimal antibody concentration for their specific research context.

What is the species reactivity profile of the antibody?

The Phospho-RUNX1 (S435) Antibody demonstrates cross-reactivity with phosphorylated RUNX1 from multiple species:

This multi-species reactivity makes the antibody valuable for comparative studies across different model systems . The antibody targets a conserved phosphorylation motif around S435 in the RUNX1 protein. The conservation of this phosphorylation site across species suggests its functional importance in RUNX1 biology and signaling pathways.

How does phosphorylation at S435 affect RUNX1 function in hematopoiesis?

Phosphorylation at S435 represents a critical post-translational modification that regulates RUNX1 function in hematopoietic development. RUNX1 forms a heterodimeric complex called Core Binding Factor (CBF) with CBFB, which is essential for normal hematopoiesis . S435 phosphorylation occurs in the C-terminus of RUNX1 and has been linked to IL-6 stimulation pathways . This phosphorylation event enhances RUNX1's interaction with KAT6A (also known as MOZ), a histone acetyltransferase that functions as a transcriptional co-activator .

In hematopoietic contexts, this enhanced interaction likely influences the transcriptional regulation of RUNX1 target genes involved in differentiation and lineage commitment. The Phospho-RUNX1 (S435) Antibody enables researchers to monitor this specific modification, providing insights into how external signals translate into altered transcriptional programs during normal hematopoiesis and pathological conditions like leukemia.

What experimental controls should be included when using Phospho-RUNX1 (S435) Antibody?

When working with phospho-specific antibodies like Phospho-RUNX1 (S435), proper experimental controls are crucial for result interpretation:

• Dephosphorylation Control: Treat a portion of your sample with lambda phosphatase to remove phosphorylations, which should eliminate signal from a truly phospho-specific antibody.

• Total RUNX1 Control: Run parallel samples with an antibody detecting total RUNX1 regardless of phosphorylation state to normalize phospho-signal to total protein levels.

• Positive Control: Include samples known to contain phosphorylated RUNX1 at S435, such as IL-6 treated cells, as IL-6 stimulation has been shown to induce phosphorylation in RUNX1's C-terminus .

• Negative Control: Include samples with conditions known to reduce or eliminate S435 phosphorylation, such as serum-starved cells or appropriate kinase inhibitor-treated samples.

• Loading Control: Include detection of housekeeping proteins to ensure equal loading across samples.

These controls help validate specificity and provide context for interpreting phosphorylation changes in experimental samples.

What sample preparation methods optimize phospho-epitope detection in Western blots?

Detecting phosphorylated proteins requires careful sample preparation to preserve phospho-epitopes. For optimal detection of Phospho-RUNX1 (S435), consider the following methodology:

• Rapid Sample Collection: Minimize the time between cell/tissue harvesting and lysis to prevent phosphatase activity.

• Phosphatase Inhibitors: Include comprehensive phosphatase inhibitor cocktails in lysis buffers (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate, and sodium pyrophosphate).

• Protease Inhibitors: Add complete protease inhibitor cocktails to prevent proteolytic degradation.

• Cold Processing: Perform all steps at 4°C to minimize enzymatic activity.

• Lysis Buffer Composition: Use a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40 or Triton X-100, 0.5% sodium deoxycholate, plus the aforementioned inhibitors.

• Sample Storage: If immediate processing is not possible, snap-freeze lysates in liquid nitrogen and store at -80°C until use.

• Gentle Thawing: Thaw samples on ice when ready to use and avoid repeated freeze-thaw cycles.

This methodical approach maximizes the preservation of phosphorylated RUNX1 for detection with the Phospho-RUNX1 (S435) Antibody .

How can Phospho-RUNX1 (S435) Antibody be used to study RUNX1 in T-cell development?

The Phospho-RUNX1 (S435) Antibody provides a powerful tool for investigating RUNX1's role in T-cell development. RUNX1 is known to control the anergy and suppressive function of regulatory T-cells (Tregs) through association with FOXP3, and it influences the expression of critical cytokines and receptors in conventional T-cells . To study these processes:

• Developmental Timeline Analysis: Track S435 phosphorylation across T-cell developmental stages using flow cytometry or Western blotting of sorted populations to identify when this modification occurs.

• Signaling Pathway Integration: Combine Phospho-RUNX1 (S435) detection with inhibitors of specific kinase pathways to determine which signaling cascades regulate this phosphorylation event during T-cell development.

• Functional Correlation: Correlate S435 phosphorylation status with functional T-cell parameters, such as cytokine production, proliferation capacity, or suppressive function.

• Co-immunoprecipitation Studies: Use the antibody to immunoprecipitate phosphorylated RUNX1 and identify differential protein interaction partners compared to non-phosphorylated RUNX1.

• Chromatin Immunoprecipitation (ChIP): Apply the antibody in ChIP experiments to determine if S435 phosphorylation affects RUNX1 binding to target genes in the T-cell lineage.

This methodological framework allows researchers to dissect how S435 phosphorylation influences RUNX1's ability to regulate genes like IL2, IFNG, TNFRSF18, IL2RA, CTLA4, and RORC in different T-cell subsets .

What kinases are responsible for RUNX1 S435 phosphorylation?

While the search results don't explicitly identify the specific kinases responsible for RUNX1 S435 phosphorylation, we can infer some possibilities based on available information. The S435 site is phosphorylated in response to IL-6 treatment , suggesting involvement of the JAK/STAT pathway or related signaling cascades activated by this cytokine. Additionally, the search results mention that HIPK2 (homeodomain-interacting protein kinase 2) phosphorylates RUNX1 at other sites (Ser-249, Thr-273, and Ser-276) when RUNX1 is associated with CBFB and DNA .

To experimentally determine the kinases responsible for S435 phosphorylation, researchers could employ:

• In vitro Kinase Assays: Test candidate kinases against recombinant RUNX1 protein or synthetic peptides containing the S435 site.

• Kinase Inhibitor Screening: Treat cells with specific kinase inhibitors and monitor effects on S435 phosphorylation using the Phospho-RUNX1 (S435) Antibody.

• Kinase Knockdown/Knockout: Deplete specific kinases using RNAi or CRISPR-based approaches and assess impact on S435 phosphorylation.

• Phosphoproteomics: Combine mass spectrometry with kinase prediction algorithms to identify potential kinases based on the amino acid sequence surrounding S435.

Understanding the responsible kinase(s) would provide insights into the signaling pathways regulating RUNX1 function in different cellular contexts.

What are common causes of weak or absent signal when using Phospho-RUNX1 (S435) Antibody?

When facing weak or absent signal with Phospho-RUNX1 (S435) Antibody, consider these methodological issues and solutions:

• Low Phosphorylation Status: RUNX1 S435 may not be phosphorylated under your experimental conditions. Consider using positive controls such as IL-6 stimulated cells .

• Phosphatase Activity: Inadequate phosphatase inhibition during sample preparation can lead to loss of phospho-epitopes. Ensure comprehensive phosphatase inhibitor cocktails are included in lysis buffers.

• Antibody Concentration: The dilution may be too high. Perform a titration experiment starting with the recommended 1:500 dilution for Western blot and adjust as needed .

• Detection System Sensitivity: Secondary antibody or detection reagents may need optimization. Consider more sensitive detection systems like enhanced chemiluminescence plus (ECL+) or fluorescent secondary antibodies.

• Protein Degradation: RUNX1 may be degraded during sample preparation. Ensure protease inhibitors are included in lysis buffers and samples are kept cold throughout processing.

• Transfer Efficiency: Poor transfer of high molecular weight proteins can occur. Optimize transfer conditions (time, buffer, membrane type) for proteins in RUNX1's size range.

• Antibody Storage: Improper storage can reduce activity. Verify the antibody has been stored at -20°C or -80°C and has not undergone multiple freeze-thaw cycles .

Systematic troubleshooting of these factors will help optimize detection of phosphorylated RUNX1.

How can I verify the specificity of Phospho-RUNX1 (S435) Antibody in my experimental system?

Verifying antibody specificity is crucial for reliable research results. For Phospho-RUNX1 (S435) Antibody, employ these methodological approaches:

• Peptide Competition Assay: Pre-incubate the antibody with excess phosphorylated and non-phosphorylated peptides containing the S435 site. Signal should be blocked by the phospho-peptide but not by the non-phospho-peptide.

• RUNX1 Knockdown/Knockout: Compare antibody reactivity in control versus RUNX1-depleted samples. Specific signal should decrease or disappear in RUNX1-depleted samples.

• Phosphatase Treatment: Treat duplicate samples with lambda phosphatase to remove phosphorylations. Signal should disappear in phosphatase-treated samples.

• Site-Directed Mutagenesis: Express wild-type RUNX1 versus S435A mutant (prevents phosphorylation). The antibody should detect wild-type but not the S435A mutant when phosphorylation is induced.

• Mass Spectrometry Validation: Perform immunoprecipitation with the antibody followed by mass spectrometry to confirm the identity of the captured protein and the presence of phosphorylation at S435.

These validation steps provide multiple lines of evidence for antibody specificity, strengthening the reliability of subsequent experimental results.

What are the considerations when analyzing RUNX1 phosphorylation in primary samples versus cell lines?

Analyzing RUNX1 phosphorylation differs significantly between primary samples and cell lines:

When working with primary samples:

• Minimize time between sample collection and processing

• Use stronger phosphatase inhibitor cocktails

• Consider cell type-specific isolation before analysis

• Include matched controls from the same donor/tissue

• Adjust lysis conditions for potentially lower protein yield

These methodological considerations help ensure that RUNX1 phosphorylation data from primary samples accurately reflects the in vivo status .

How can Phospho-RUNX1 (S435) Antibody be used to study hematological malignancies?

Phospho-RUNX1 (S435) Antibody offers valuable research applications in studying hematological malignancies, particularly those involving RUNX1 dysregulation:

• Diagnostic Biomarker Research: Investigate whether S435 phosphorylation status correlates with specific leukemia subtypes, disease progression, or treatment response.

• Therapeutic Target Validation: Assess how existing or experimental drugs affect RUNX1 S435 phosphorylation in malignant cells, potentially identifying new therapeutic mechanisms.

• Signaling Pathway Analysis: Map altered signaling networks in leukemia by examining how S435 phosphorylation changes in response to cytokines or growth factors in malignant versus normal hematopoietic cells.

• Fusion Protein Studies: For leukemias involving RUNX1 fusion proteins (e.g., RUNX1-ETO in t(8;21) AML), determine if S435 phosphorylation is preserved and how it may affect fusion protein function.

• Drug Resistance Mechanisms: Investigate whether changes in RUNX1 phosphorylation correlate with resistance to standard therapies in patient samples or model systems.

The antibody's ability to work in both human and mouse samples enables translational research spanning from mouse models to patient specimens, creating a comprehensive research pipeline for understanding RUNX1 phosphorylation in hematological malignancies.

What is the relationship between RUNX1 S435 phosphorylation and other post-translational modifications?

RUNX1 undergoes multiple post-translational modifications that collectively regulate its activity, stability, and interactions. The relationship between S435 phosphorylation and other modifications represents an important research direction:

• Phosphorylation Crosstalk: S435 phosphorylation may influence or be influenced by other phosphorylation events on RUNX1. The search results indicate that RUNX1 is also phosphorylated at Ser-249, Thr-273, and Ser-276 by HIPK2 when associated with CBFB and DNA . These phosphorylation events promote subsequent EP300 phosphorylation . Researchers can use Phospho-RUNX1 (S435) Antibody in combination with antibodies against other phosphorylation sites to map phosphorylation patterns and their interdependencies.

• Methylation Interaction: RUNX1 is known to be methylated , and this modification may work in concert with phosphorylation to fine-tune RUNX1 function. Researchers can investigate whether S435 phosphorylation affects methylation patterns or vice versa.

• Acetylation Coordination: S435 phosphorylation enhances interaction with KAT6A , which may influence acetylation of RUNX1 or associated histones. This interaction can be studied using co-immunoprecipitation experiments with Phospho-RUNX1 (S435) Antibody.

• Ubiquitination and Stability: Researchers can investigate whether S435 phosphorylation affects RUNX1 ubiquitination and protein stability, potentially linking this phosphorylation event to protein turnover regulation.

Understanding these modification relationships will provide a more complete picture of how RUNX1 activity is regulated in normal development and disease states.

How can Phospho-RUNX1 (S435) Antibody be integrated with other research technologies?

Integration of Phospho-RUNX1 (S435) Antibody with advanced research technologies creates powerful experimental approaches:

• Single-Cell Analysis: Combine with single-cell Western blot or mass cytometry (CyTOF) to examine S435 phosphorylation heterogeneity within hematopoietic populations.

• Spatial Transcriptomics: Integrate immunofluorescence using Phospho-RUNX1 (S435) Antibody with spatial transcriptomics to correlate S435 phosphorylation with gene expression patterns in tissue contexts.

• CRISPR Screens: Use the antibody as a readout in CRISPR screens to identify genes regulating S435 phosphorylation.

• Proximity Ligation Assays: Combine with antibodies against potential interaction partners to visualize and quantify protein interactions that depend on S435 phosphorylation.

• ChIP-seq Integration: Use in parallel with ChIP-seq to correlate genome-wide binding patterns with phosphorylation status.

• Organoid Systems: Apply the antibody in organoid cultures to study RUNX1 phosphorylation in three-dimensional tissue contexts that better mimic in vivo conditions.

• Live-Cell Imaging: Develop complementary fluorescent biosensors for RUNX1 phosphorylation that can be validated using the antibody, enabling dynamic studies of phosphorylation events.

These integrative approaches extend the utility of the antibody beyond traditional applications like Western blot and ELISA , creating new opportunities for understanding RUNX1 biology.