Phospho-RUNX1 (Ser276) Antibody

What is the functional significance of RUNX1 phosphorylation at Ser276?

Further research suggests phosphorylation at Ser276 along with other serine residues may reduce interactions with histone deacetylases (HDAC1/HDAC3), thereby potentially increasing transcriptional activity . Phosphorylation at this site may be regulated by cyclin-dependent kinases and appears to be involved in cell cycle control mechanisms in hematopoietic development .

What applications are Phospho-RUNX1 (Ser276) antibodies suitable for?

Phospho-RUNX1 (Ser276) antibodies are validated for multiple research applications:

These recommendations are consistent across multiple antibody suppliers , suggesting reliable performance across these applications when used within the specified dilution ranges.

What is the specificity of Phospho-RUNX1 (Ser276) antibodies?

These antibodies are designed to detect endogenous levels of RUNX1 protein only when phosphorylated at Ser276 . Specificity is typically achieved through immunogen design - the antibodies are produced against synthesized peptides derived from human AML1 (RUNX1) surrounding the phosphorylation site, with the amino acid range typically specified as 269-318 .

The specificity can be confirmed through various controls, including the use of phosphatase treatment or comparison with antibodies recognizing total RUNX1 regardless of phosphorylation status. The modification sequence recognized is typically "PIsPG" (where "s" represents the phosphorylated serine) .

How should samples be prepared to preserve RUNX1 Ser276 phosphorylation status?

Phosphorylation states are notoriously labile during sample preparation. For optimal detection:

• Include phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate) in all lysis and wash buffers.

• Perform cell lysis and protein extraction rapidly at 4°C to minimize dephosphorylation.

• For cell culture experiments investigating RUNX1 phosphorylation, consider serum starvation followed by stimulation protocols to synchronize phosphorylation events.

• When fixing tissues for IHC or IF applications, minimize the fixation time as prolonged fixation may mask phospho-epitopes.

• For Western blotting, use freshly prepared samples whenever possible. If storage is necessary, snap-freeze lysates and store at -80°C with phosphatase inhibitors.

Similar methodologies have been employed in studies examining RUNX1 phosphorylation status, such as those investigating RUNX1 phosphorylation in leukemogenesis models .

What positive and negative controls should be used when working with Phospho-RUNX1 (Ser276) antibodies?

For rigorous experimental design, the following controls are recommended:

Positive Controls:

• Cell lines known to express phosphorylated RUNX1 (e.g., hematopoietic cell lines like Ba/F3)

• Cells treated with phosphatase inhibitors

• Cells synchronized to cell cycle phases where RUNX1 phosphorylation is elevated

Negative Controls:

• Phosphatase-treated samples (lambda phosphatase)

• RUNX1 knockout or knockdown cells

• Non-hematopoietic cells with low RUNX1 expression (e.g., certain brain or heart-derived cell lines)

• Blocking peptide competition assays using the phospho-peptide immunogen

What are the recommended storage conditions for Phospho-RUNX1 (Ser276) antibodies?

For optimal antibody performance and shelf-life:

• Store at -20°C (typically -15°C to -25°C range, but do not store below -25°C)

• Most commercial antibodies are formulated in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• Avoid repeated freeze-thaw cycles

• Typical shelf life is up to 1 year when stored properly

• Aliquot antibodies upon first thawing to minimize freeze-thaw cycles

How can Phospho-RUNX1 (Ser276) antibodies be used to study RUNX1 interactions with transcriptional co-regulators?

Research has shown that phosphorylation of RUNX1 at Ser276 (among other serine residues) affects its interactions with transcriptional co-regulators, particularly histone deacetylases (HDACs). To study these interactions:

• Co-immunoprecipitation (Co-IP) assays: Immunoprecipitate with either Phospho-RUNX1 (Ser276) antibody or antibodies against potential interacting partners (e.g., HDAC1, HDAC3) followed by Western blotting to detect the reciprocal protein .

• Chromatin immunoprecipitation (ChIP) assays: Use Phospho-RUNX1 (Ser276) antibodies to determine if phosphorylated RUNX1 differentially associates with specific genomic loci compared to total RUNX1.

• Sequential ChIP (Re-ChIP): Perform initial ChIP with Phospho-RUNX1 (Ser276) antibody followed by a second round of immunoprecipitation with antibodies against co-regulators to identify co-occupancy at specific genomic regions.

• Proximity ligation assays (PLA): Visualize and quantify interactions between phosphorylated RUNX1 and potential binding partners in situ within cells.

Studies have demonstrated that mutation of serine residues (including Ser276-equivalent) to aspartic acid (phosphomimetic) reduced interaction with HDAC1 or HDAC3 approximately 4-fold, suggesting phosphorylation weakens these interactions .

How does cell cycle progression influence RUNX1 Ser276 phosphorylation?

RUNX1 is regulated in a cell cycle-dependent manner, with levels increasing as hematopoietic cells progress from G1 to S and from S to G2/M . To study cell cycle-dependent phosphorylation of RUNX1 at Ser276:

• Cell synchronization protocols: Utilize methods such as double thymidine block, nocodazole treatment, or serum starvation/refeeding to synchronize cells at specific cell cycle stages.

• Flow cytometry with phospho-specific antibodies: Perform intracellular staining with Phospho-RUNX1 (Ser276) antibodies combined with DNA content analysis to correlate phosphorylation status with cell cycle phase.

• Kinase inhibition studies: Use specific inhibitors of cyclin-dependent kinases (CDKs) to determine which kinases regulate Ser276 phosphorylation during different cell cycle phases.

Evidence suggests that CDKs (particularly cdk1/cyclin B and cdk6/cyclin D3) are responsible for phosphorylating RUNX1 at various serine residues, and that this phosphorylation can influence RUNX1 stability and transcriptional activity through the cell cycle .

What is the relationship between RUNX1 Ser276 phosphorylation and leukemogenesis?

The role of RUNX1 phosphorylation in leukemia development is an active area of research:

• Analysis of patient samples: Compare phosphorylation status of RUNX1 at Ser276 between normal hematopoietic cells and leukemic blasts using Phospho-RUNX1 (Ser276) antibodies in Western blot, IHC, or flow cytometry applications.

• Leukemia model systems: Investigate whether expression of leukemia-associated fusion proteins (e.g., CBFβ-SMMHC) affects RUNX1 Ser276 phosphorylation status .

• Mutagenesis studies: Utilize phosphomimetic (S→D) or phospho-deficient (S→A) RUNX1 mutants to assess functional consequences on hematopoietic differentiation and transformation.

Research has suggested that retention of RUNX1 phosphorylation may be one mechanism for accelerated leukemogenesis in certain contexts, such as with truncated CBFβ-SMMHC fusion proteins . Additionally, the interaction between phosphorylated RUNX1 and the p300 histone acetyltransferase appears to play a role in leukemic transformation .

Why might Western blot detection of phosphorylated RUNX1 (Ser276) show weak signal?

Several factors can contribute to weak signal when detecting phosphorylated RUNX1:

• Low abundance of phosphorylated form: Phosphorylation is often a transient modification affecting only a fraction of the total protein pool. Consider enrichment strategies like immunoprecipitation before Western blotting.

• Rapid dephosphorylation: Ensure phosphatase inhibitors are fresh and present at sufficient concentrations in all buffers.

• Antibody sensitivity: The observed molecular weight band for RUNX1 is approximately 55kD . Ensure loading sufficient protein (typically 20-50μg of total protein) and optimize primary antibody concentration within recommended ranges (1:500-1:2000) .

• Detection system limitations: Consider using enhanced chemiluminescence detection systems with extended exposure times or switch to more sensitive detection methods like fluorescent secondary antibodies.

• Epitope masking: Some sample preparation methods may cause conformational changes that mask the phospho-epitope. Try alternative extraction methods or gentler denaturation conditions.

How can specificity of Phospho-RUNX1 (Ser276) antibodies be verified in experimental systems?

To confirm antibody specificity:

• Peptide competition assays: Pre-incubate antibody with excess phosphorylated peptide immunogen before use in applications. Specific signal should be abolished.

• Phosphatase treatment: Treat half of your sample with lambda phosphatase before immunoblotting. The signal should be eliminated or significantly reduced in the treated sample.

• Genetic validation: Use RUNX1 knockout/knockdown cells or cells expressing phospho-deficient RUNX1 mutants (S276A) as negative controls.

• Cross-reactivity assessment: Test antibody against recombinant phosphorylated and non-phosphorylated RUNX1 proteins to confirm phospho-specificity.

• Multiple antibody validation: When possible, confirm results using alternative antibodies against the same phosphorylation site from different suppliers or different clones.

What are the common pitfalls when using Phospho-RUNX1 (Ser276) antibodies in immunofluorescence applications?

When using these antibodies for immunofluorescence:

• Fixation-induced epitope masking: Phospho-epitopes can be sensitive to fixation. Compare paraformaldehyde, methanol, and acetone fixation to determine optimal conditions.

• Autofluorescence interference: Particularly in tissues with high heme content (like bone marrow), autofluorescence can mask specific signals. Consider using Sudan Black B treatment or spectral unmixing techniques.

• Background from non-specific binding: Optimize blocking conditions (typically 1-5% BSA or normal serum matching secondary antibody host) and antibody dilutions (starting at 1:50-1:200) .

• Nuclear antigen accessibility: For better nuclear antigen detection, include a permeabilization step with 0.1-0.5% Triton X-100.

• Signal amplification needs: For low abundance phospho-proteins, consider tyramide signal amplification or similar techniques to enhance detection sensitivity.

How can phosphorylation at Ser276 be integrated with other RUNX1 post-translational modifications?

RUNX1 undergoes multiple post-translational modifications that collectively determine its function. To study these in an integrated manner:

• Sequential immunoprecipitation: First immunoprecipitate with Phospho-RUNX1 (Ser276) antibody, then probe for other modifications (methylation, acetylation, ubiquitination) or vice versa.

• Mass spectrometry analysis: Use phospho-enrichment followed by mass spectrometry to identify co-occurring modifications on RUNX1 molecules phosphorylated at Ser276.

• Modification-specific antibody arrays: Probe multiple RUNX1 modifications simultaneously using antibody arrays specific for different post-translational modifications.

Research indicates that RUNX1 undergoes multiple phosphorylation events (Ser249, Ser266, Ser276) , and that phosphorylation may influence subsequent modifications. For instance, phosphorylation enhances interaction with KAT6A and can promote subsequent EP300 phosphorylation .

What experimental approaches can resolve contradictions between in vitro and in vivo studies of RUNX1 Ser276 phosphorylation?

The contradictions between transfection assays (suggesting phosphorylation enhances RUNX1 activity) and knock-in mouse studies (showing normal function despite lack of phosphorylation) highlight important experimental considerations:

• Cell-type specific effects: Study phosphorylation in relevant hematopoietic cell types rather than heterologous expression systems.

• Developmental timing analysis: Examine phosphorylation status throughout developmental processes using developmental time-course studies.

• Physiological expression levels: Ensure experimental systems maintain physiological RUNX1 levels rather than overexpression, which may bypass regulatory mechanisms.

• Compensatory mechanism identification: Use phosphoproteomics to identify potential compensatory phosphorylation events that may occur in knock-in models.

• Contextual dependency assessment: Study phosphorylation under various stimuli (cytokines, stress conditions, differentiation agents) to identify context-dependent functions.

These approaches can help reconcile seemingly contradictory findings by providing a more nuanced understanding of RUNX1 regulation under physiologically relevant conditions.

How might single-cell analysis techniques advance our understanding of RUNX1 Ser276 phosphorylation?

Single-cell methodologies offer promising avenues for studying RUNX1 phosphorylation heterogeneity:

• Single-cell mass cytometry (CyTOF): Develop metal-conjugated Phospho-RUNX1 (Ser276) antibodies for use in mass cytometry to correlate phosphorylation status with lineage markers and other signaling events at single-cell resolution.

• Imaging mass cytometry: Apply similar approaches in tissue contexts to understand spatial relationships between cells with different RUNX1 phosphorylation states.

• Single-cell Western blotting: Adapt emerging single-cell Western blotting technologies to quantify phosphorylation status in individual cells.

• Live-cell imaging with phospho-sensors: Develop FRET-based sensors that can report on RUNX1 Ser276 phosphorylation in living cells in real-time.

These approaches would be particularly valuable for understanding the dynamics of RUNX1 phosphorylation during hematopoietic differentiation and in leukemic transformation, where cellular heterogeneity is a significant factor.

What are the therapeutic implications of targeting pathways that regulate RUNX1 Ser276 phosphorylation?

Understanding RUNX1 phosphorylation mechanisms opens several therapeutic avenues:

• CDK inhibitor applications: Since cyclin-dependent kinases appear to regulate RUNX1 phosphorylation , evaluate the effects of selective CDK inhibitors on RUNX1 function in leukemia models.

• Phosphatase modulator development: Identify phosphatases that dephosphorylate RUNX1 at Ser276 and assess whether modulating their activity offers therapeutic benefit.

• Disruption of phosphorylation-dependent interactions: Develop small molecules or peptide mimetics that specifically disrupt or enhance interactions between phosphorylated RUNX1 and its binding partners.

• Combination therapy strategies: Investigate whether modulating RUNX1 phosphorylation status sensitizes leukemic cells to conventional chemotherapeutics.

• Biomarker potential: Evaluate whether RUNX1 Ser276 phosphorylation status can serve as a biomarker for disease progression or treatment response in hematological malignancies.

These approaches could potentially lead to new therapeutic strategies for leukemias with dysregulated RUNX1 function, particularly in contexts where RUNX1 mutation or chromosomal translocations occur .

What are the key differences between polyclonal and monoclonal Phospho-RUNX1 (Ser276) antibodies?

Both polyclonal and monoclonal antibodies targeting Phospho-RUNX1 (Ser276) are commercially available, each with distinct characteristics:

The choice between polyclonal and monoclonal antibodies should be guided by the specific research application, with polyclonals potentially offering higher sensitivity and monoclonals providing greater specificity and reproducibility.

How does the immunogen design influence antibody performance for detecting Phospho-RUNX1 (Ser276)?

The immunogen design is critical for phospho-specific antibody performance:

• Peptide length: The immunogen peptide length (typically spanning residues 269-318) provides context around the phosphorylation site, which may influence antibody specificity and sensitivity.

• Carrier protein conjugation: Most phospho-peptides are conjugated to carrier proteins (typically KLH or BSA) for immunization, which can affect antibody generation efficiency.

• Phosphorylation site position: The position of Ser276 within the immunogen (whether centrally located or near the terminus) affects antibody accessibility to the phospho-epitope.

• Surrounding sequence conservation: The high conservation of the sequence surrounding Ser276 across species explains the cross-reactivity of these antibodies with human, mouse, and rat samples .

• Structural considerations: The three-dimensional presentation of the phospho-epitope in the native protein versus the immunizing peptide can affect antibody recognition in applications where protein folding is preserved.

Understanding these factors can help researchers select the most appropriate antibody for their specific experimental conditions and interpret results accurately.