RUNX1 (Ab-435) Antibody

What is RUNX1 and what are its primary functions in hematopoiesis?

RUNX1, also known as AML1 or CBFA2, is a transcription factor encoded by a gene located at chromosome position 21q22.3 and belongs to the RUNX transcription factor family. The protein contains a Runt homology domain (RHD) that mediates the formation of heterodimers with Core-Binding Factor β (CBFβ) and promotes DNA binding to regulate the expression of multiple genes . RUNX1 is essential for normal hematopoiesis and plays critical roles in various biological processes including cell differentiation, proliferation, cell cycle regulation, DNA repair, and apoptosis .

The functional significance of RUNX1 has been demonstrated in mouse models where RUNX1 knockout during embryonic development leads to complete failure of hematopoietic stem cell (HSC) production . RUNX1 forms the heterodimeric complex core-binding factor (CBF) with CBFΒ, recognizing the core consensus binding sequence 5'-TGTGGT-3', or rarely, 5'-TGCGGT-3', within regulatory regions of target genes . This complex binds to enhancers and promoters of various genes, including murine leukemia virus, polyomavirus enhancer, T-cell receptor enhancers, and the promoters of LCK, IL3, and GM-CSF .

What is RUNX1 (Ab-435) Antibody and what is its specificity?

RUNX1 (Ab-435) Antibody, also referenced as Anti-Phospho-RUNX1-Ser435 antibody, is a rabbit polyclonal antibody that specifically detects RUNX1 protein only when phosphorylated at serine 435 . This antibody was generated using a synthesized peptide derived from human AML1 around the phosphorylation site of Ser435, specifically targeting the amino acid range 401-450 . It is affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogen .

The antibody's specificity for the phosphorylated form of RUNX1 makes it particularly valuable for studying post-translational modifications of RUNX1 that may regulate its function. The antibody recognizes phosphorylated RUNX1 in human, mouse, and rat samples, making it applicable across multiple model systems . Understanding the phosphorylation status of RUNX1 at Ser435 can provide insights into how this modification affects RUNX1's interaction with other proteins and its transcriptional activity.

What is the significance of Ser435 phosphorylation in RUNX1 function?

Phosphorylation at Ser435 is one of several post-translational modifications that regulate RUNX1 activity. While RUNX1 undergoes phosphorylation at multiple sites, including Ser249, Thr273, and Ser276 by homeodomain-interacting protein kinase 2 (HIPK2) when associated with CBFB and DNA, the specific phosphorylation at Ser435 appears to have distinct regulatory functions .

Phosphorylation of RUNX1 in its C-terminus, which includes Ser435, can be induced by IL-6 treatment. This phosphorylation enhances RUNX1's interaction with KAT6A (also known as MOZ or MYST3), a histone acetyltransferase . The phosphorylation of RUNX1 promotes subsequent EP300 (p300) phosphorylation, suggesting a cascade of phosphorylation events that regulate transcriptional activity . The site-specific phosphorylation of RUNX1 may represent a mechanism for fine-tuning its transcriptional function in different cellular contexts and in response to various signaling pathways.

What are the validated applications for RUNX1 (Ab-435) Antibody?

The RUNX1 (Ab-435) Antibody has been validated for several research applications, with the primary validated uses being Western Blot (WB) and Enzyme-Linked Immunosorbent Assay (ELISA) . The recommended dilution ranges for these applications are 1:500-1:2000 for Western Blot and 1:10000 for ELISA . This information is crucial for researchers planning experiments to ensure optimal antibody performance.

The antibody is provided in liquid form in PBS containing 50% Glycerol, 0.5% BSA, and 0.02% Sodium Azide, with a concentration of 1 mg/mL . It should be stored at -20°C for up to 1 year from the date of receipt, and repeated freeze-thaw cycles should be avoided to maintain antibody integrity and performance . These storage conditions are important for preserving antibody activity over time.

What is the optimal protocol for using RUNX1 (Ab-435) Antibody in Western Blot analysis?

For Western Blot analysis using RUNX1 (Ab-435) Antibody, researchers should follow these methodological steps:

• Sample Preparation: Extract proteins from cells or tissues of interest. Based on validated data, extracts from P19, HeLa, and 293 cells have been successfully used with RUNX1 antibodies .

• Protein Separation: Use SDS-PAGE for protein separation. A discontinuous SDS-PAGE system with 5% enrichment gel and 15% separation gel (Tris-Glycine) has been validated for RUNX1 analysis .

• Protein Transfer: Transfer separated proteins to a suitable membrane (PVDF or nitrocellulose).

• Blocking: Block the membrane with appropriate blocking buffer to prevent non-specific binding.

• Primary Antibody Incubation: Dilute RUNX1 (Ab-435) Antibody at 1:500-1:2000 in antibody dilution buffer and incubate the membrane .

• Washing: Wash the membrane thoroughly to remove unbound primary antibody.

• Secondary Antibody Incubation: Incubate with an appropriate HRP-conjugated secondary antibody against rabbit IgG.

• Detection: Use an enhanced chemiluminescence (ECL) detection system to visualize the results.

Researchers should include positive controls (samples known to contain phosphorylated RUNX1 at Ser435) and negative controls (samples lacking phosphorylated RUNX1 or treated with phosphatase) to validate the specificity of the signal.

How can researchers ensure specificity when using RUNX1 (Ab-435) Antibody?

To ensure specificity when using RUNX1 (Ab-435) Antibody, researchers should implement several validation strategies:

• Phosphatase Controls: Treat duplicate samples with lambda phosphatase to remove phosphorylation. The antibody should not detect RUNX1 in dephosphorylated samples if it is truly specific for the phosphorylated form.

• Peptide Competition Assay: Pre-incubate the antibody with the phosphorylated peptide used as the immunogen (derived from the region around Ser435) before application to samples. This should abolish specific binding.

• Knockout/Knockdown Validation: Use RUNX1 knockout or knockdown samples as negative controls to confirm the specificity of the detected band.

• Cross-Validation: Compare results with other phospho-specific antibodies or with general RUNX1 antibodies after immunoprecipitation and phosphatase treatment.

• Multiple Detection Methods: Confirm phosphorylation using alternative methods such as mass spectrometry.

• Stimulation Experiments: Use treatments known to induce or reduce RUNX1 phosphorylation at Ser435 (e.g., IL-6 treatment has been shown to induce C-terminal phosphorylation of RUNX1) .

These validation steps are crucial for ensuring that experimental results accurately reflect the phosphorylation state of RUNX1 at Ser435.

How can RUNX1 (Ab-435) Antibody be used to study RUNX1 phosphorylation in hematological malignancies?

RUNX1 (Ab-435) Antibody can be utilized in several sophisticated approaches to investigate RUNX1 phosphorylation in hematological malignancies:

• Comparative Phosphorylation Analysis: Researchers can compare the levels of Ser435 phosphorylation between normal hematopoietic cells and malignant cells using Western blot or ELISA. Mutations in the RUNX1 gene are frequently found in various hematological tumors, particularly myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and are associated with poor prognosis .

• Signaling Pathway Investigation: The antibody can be used to determine how various signaling pathways affect RUNX1 phosphorylation at Ser435 in malignant cells. For instance, researchers can examine whether abnormal activation of upstream kinases contributes to altered RUNX1 phosphorylation and function.

• Therapeutic Response Monitoring: Changes in RUNX1 Ser435 phosphorylation in response to therapeutic agents can be monitored to understand drug mechanisms and identify potential biomarkers of treatment response.

• Correlation with Disease Progression: By analyzing RUNX1 Ser435 phosphorylation in patient samples at different disease stages, researchers can investigate whether this modification correlates with disease progression or clinical outcomes.

• Investigation of RUNX1 Fusion Proteins: In cases where RUNX1 is involved in chromosomal translocations, such as t(8;21)(q22;q22.1) leading to RUNX1-RUNX1T1 fusion , the antibody can help determine whether the fusion protein is still phosphorylated at Ser435 and how this affects its function.

What role does RUNX1 Ser435 phosphorylation play in the interaction with transcriptional cofactors?

Phosphorylation of RUNX1 at Ser435 affects its interaction with transcriptional cofactors, which is crucial for understanding its functional regulation:

• Enhanced KAT6A Interaction: Phosphorylation in RUNX1's C-terminus (including Ser435) upon IL-6 treatment enhances its interaction with KAT6A (MOZ/MYST3), a histone acetyltransferase . This interaction may alter chromatin accessibility and gene expression patterns.

• EP300 Phosphorylation Cascade: RUNX1 phosphorylation promotes subsequent phosphorylation of EP300 (p300) , a histone acetyltransferase that functions as a transcriptional coactivator. This suggests a phosphorylation cascade that regulates transcriptional activation.

• CBFβ Binding Regulation: While the primary interaction between RUNX1 and CBFβ is mediated through the Runt homology domain (RHD), phosphorylation at Ser435 might indirectly affect this interaction or the DNA-binding capacity of the heterodimeric complex.

• Transcriptional Complex Formation: RUNX1 (Ab-435) Antibody can be used in co-immunoprecipitation experiments to identify proteins that preferentially interact with phosphorylated RUNX1 compared to the non-phosphorylated form.

• Chromatin Immunoprecipitation (ChIP) Analysis: The antibody can be employed in ChIP experiments to determine whether Ser435 phosphorylation affects RUNX1 binding to specific genomic loci and recruitment of transcriptional cofactors.

What are common challenges when using phospho-specific antibodies like RUNX1 (Ab-435) Antibody?

Working with phospho-specific antibodies like RUNX1 (Ab-435) Antibody presents several technical challenges:

• Phosphatase Activity: Endogenous phosphatases in cell and tissue lysates can dephosphorylate RUNX1 during sample preparation. Researchers should include phosphatase inhibitors in lysis buffers and maintain samples at cold temperatures to minimize this issue.

• Epitope Masking: Protein-protein interactions or additional post-translational modifications near Ser435 might mask the epitope, preventing antibody binding. Different sample preparation methods (denaturing vs. native conditions) should be tested.

• Cross-Reactivity: Phospho-specific antibodies may sometimes recognize similar phosphorylated motifs in other proteins. Careful validation using appropriate controls (as described in section 2.3) is essential.

• Low Abundance: Phosphorylated forms of proteins often exist at lower abundance than their non-phosphorylated counterparts. Enrichment strategies such as immunoprecipitation before Western blotting may be necessary for detection.

• Signal Variability: The phosphorylation status of RUNX1 may change rapidly in response to cellular conditions or experimental manipulations. Standardized sample collection and preparation protocols are crucial for reproducible results.

How can researchers interpret contradictory results between RUNX1 (Ab-435) Antibody and other RUNX1 antibodies?

When faced with contradictory results between RUNX1 (Ab-435) Antibody and other RUNX1 antibodies, researchers should consider these analytical approaches:

• Epitope Differences: RUNX1 (Ab-435) Antibody recognizes only the phosphorylated form at Ser435, while general RUNX1 antibodies detect total RUNX1 protein regardless of phosphorylation status. Different signal patterns are expected and should be interpreted in this context.

• Isoform Specificity: RUNX1 has multiple isoforms (e.g., AML-1G, AML-1L) with different functions . Some antibodies may preferentially detect certain isoforms. Researchers should identify which isoforms contain the Ser435 phosphorylation site.

• Technical Verification: When discrepancies occur, researchers should verify whether the phospho-specific signal disappears after phosphatase treatment, which would confirm its specificity.

• Cellular Context: Phosphorylation status depends on cellular context, activation state, and signaling conditions. Different results may reflect biological differences rather than technical issues.

• Correlation Analysis: Quantify the relationship between total RUNX1 and phosphorylated RUNX1 across multiple samples to identify patterns that might explain discrepancies.

• Multiple Detection Methods: Use complementary methods (e.g., Phos-tag SDS-PAGE, mass spectrometry) to verify phosphorylation status and resolve contradictions.

How can RUNX1 (Ab-435) Antibody be used to study leukemogenic RUNX1 fusion proteins?

RUNX1 (Ab-435) Antibody offers valuable approaches for studying leukemogenic RUNX1 fusion proteins:

• Phosphorylation Status of Fusion Proteins: Researchers can investigate whether RUNX1 fusion proteins, such as RUNX1-RUNX1T1 resulting from t(8;21)(q22;q22.1) translocation , retain the Ser435 phosphorylation site and whether this site is still phosphorylated in the fusion context.

• Altered Regulation: By comparing the phosphorylation patterns between wild-type RUNX1 and fusion proteins under various conditions, researchers can gain insights into how the fusion alters normal RUNX1 regulation.

• Functional Significance: Using site-directed mutagenesis to create phosphomimetic (S435D/E) or phospho-deficient (S435A) variants of RUNX1 fusion proteins, researchers can assess the functional significance of this phosphorylation site in leukemogenesis.

• Therapeutic Targeting: The antibody can help evaluate whether drugs targeting signaling pathways that regulate RUNX1 phosphorylation affect the function of RUNX1 fusion proteins, potentially identifying therapeutic vulnerabilities.

• Patient Sample Analysis: Examining the phosphorylation status of RUNX1 and its fusion proteins in patient samples may reveal correlations with disease characteristics, treatment response, or prognosis.

What insights can RUNX1 (Ab-435) Antibody provide about RUNX1's role in normal hematopoietic development?

RUNX1 (Ab-435) Antibody can help elucidate RUNX1's role in normal hematopoietic development through these research approaches:

• Developmental Phosphorylation Patterns: By analyzing the phosphorylation of RUNX1 at Ser435 during different stages of hematopoietic development, researchers can identify when this modification occurs and correlate it with specific developmental events.

• Lineage-Specific Regulation: RUNX1 is involved in lineage commitment of immature T cell precursors and plays roles in various hematopoietic lineages . The antibody can help determine whether Ser435 phosphorylation varies across different hematopoietic lineages, potentially contributing to lineage-specific functions.

• Regulatory Interactions: RUNX1 controls the anergy and suppressive function of regulatory T-cells by associating with FOXP3, activates IL2 and IFNG expression, and downregulates TNFRSF18, IL2RA, and CTLA4 in conventional T-cells . The antibody can help determine if Ser435 phosphorylation affects these regulatory interactions.

• Differentiation Signals: Since RUNX1 knockout during embryonic development leads to complete failure of HSC production , researchers can examine how signaling pathways that regulate hematopoietic differentiation affect RUNX1 Ser435 phosphorylation.

• Temporal Dynamics: Using the antibody in time-course experiments during differentiation protocols can reveal the temporal dynamics of RUNX1 phosphorylation in relation to key developmental transitions.

This table summarizes the known phosphorylation sites of RUNX1, their associated kinases, functional significance, and detection methods based on the provided search results .

How does the phosphorylation state of RUNX1 contribute to its tumor suppressor function?

The phosphorylation state of RUNX1, including at Ser435, may contribute to its tumor suppressor function through several mechanisms:

• Transcriptional Regulation: RUNX1 regulates genes involved in cell cycle, apoptosis, and differentiation . Phosphorylation at Ser435 might modulate its transcriptional activity on these target genes, affecting cell growth and survival pathways.

• Protein Stability and Degradation: Phosphorylation can influence protein stability by affecting recognition by the ubiquitin-proteasome system. Altered phosphorylation could lead to abnormal RUNX1 protein levels in cancer cells.

• Protein-Protein Interactions: Phosphorylation at Ser435 enhances interaction with KAT6A , which might be important for RUNX1's tumor suppressor function. Disruption of this interaction through mutations or altered phosphorylation could contribute to leukemogenesis.

• DNA Damage Response: RUNX1 is involved in DNA repair processes . Phosphorylation might regulate its recruitment to sites of DNA damage or interaction with DNA repair machinery.

• Genomic Stability: Loss of RUNX1 function through mutations or altered post-translational modifications is associated with genomic instability, a hallmark of cancer. Investigating how Ser435 phosphorylation affects genomic stability could provide insights into RUNX1's tumor suppressor mechanisms.