Phospho-RUNX1 (S249) Antibody

What is the Phospho-RUNX1 (S249) Antibody and what does it specifically detect?

The Phospho-RUNX1 (S249) Antibody is a rabbit polyclonal antibody that specifically recognizes the RUNX1 protein (also known as AML1 or CBFA2) only when phosphorylated at serine 249. This antibody does not detect non-phosphorylated RUNX1 or phosphorylation at other sites, making it valuable for studying specific post-translational modifications of RUNX1 . The specificity for phosphorylated Ser249 allows researchers to monitor this particular phosphorylation event, which is associated with RUNX1 activation and function as a transcription factor.

What are the primary research applications for this antibody?

The primary research applications for Phospho-RUNX1 (S249) Antibody include:

• Western Blot (WB): Most commonly used at dilutions ranging from 1:500 to 1:2000

• Enzyme-Linked Immunosorbent Assay (ELISA): Typically used at dilutions around 1:5000

• Immunohistochemistry (IHC): Supported by some manufacturers

• Immunofluorescence/Immunocytochemistry (IF/ICC): Applicable for cellular localization studies

Each application requires appropriate optimization, and researchers should conduct preliminary experiments to determine optimal working concentrations for their specific experimental systems.

Which species does this antibody recognize?

The Phospho-RUNX1 (S249) Antibody demonstrates cross-reactivity with:

• Human RUNX1

• Mouse RUNX1

• Rat RUNX1

Some manufacturers also report reactivity with monkey samples . Several antibody sources predict potential cross-reactivity with bovine, horse, sheep, rabbit, dog, chicken, and Xenopus, though these applications would require validation by the researcher before use in critical experiments .

How should the antibody be stored and handled to maintain its activity?

For optimal antibody performance and stability:

• Store at -20°C for long-term storage (up to 1 year from receipt)

• For frequent use and short-term storage, some manufacturers recommend 4°C for up to one month

• Avoid repeated freeze-thaw cycles as this can degrade antibody quality

• The antibody is typically supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• Concentration is generally 1 mg/mL

Following these storage guidelines will help maintain antibody specificity and sensitivity for research applications.

What is the recommended protocol for Western blot using this antibody?

A general protocol for Western blot using Phospho-RUNX1 (S249) Antibody includes:

• Sample preparation: Prepare protein lysates from tissues or cells of interest with phosphatase inhibitors to preserve phosphorylation states

• Protein separation: Run 20-50 μg of protein on SDS-PAGE (typically 10-12%)

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane

• Blocking: Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation: Dilute Phospho-RUNX1 (S249) Antibody 1:500-1:2000 in blocking buffer and incubate overnight at 4°C

• Washing: Wash membrane 3-5 times with TBST

• Secondary antibody: Incubate with HRP-conjugated anti-rabbit IgG (1:5000-1:10000) for 1 hour at room temperature

• Washing: Wash membrane 3-5 times with TBST

• Detection: Develop using ECL detection reagents

• Analysis: The expected molecular weight of RUNX1 is approximately 49 kDa, though bands at 55 kDa and 70 kDa may also be observed due to post-translational modifications or splice variants

Researchers should optimize this protocol for their specific experimental conditions.

What controls should be included when using the Phospho-RUNX1 (S249) Antibody?

For rigorous experimental design, include the following controls:

• Positive control: Lysates from cells/tissues known to express phosphorylated RUNX1 (S249), such as:

  • Hematopoietic cells treated with IL-6

  • Cells treated with agents that activate MAPK/ERK signaling

• Negative controls:

  • Lysates from cells treated with lambda phosphatase to remove phosphorylation

  • Samples from tissues that do not express RUNX1 (e.g., brain tissue)

  • Competitive blocking with the immunizing peptide

• Antibody controls:

  • Omission of primary antibody

  • Isotype control (rabbit IgG at equivalent concentration)

  • Use of a pan-RUNX1 antibody on parallel samples to assess total RUNX1 levels

These controls help validate specific detection of phosphorylated RUNX1 at Ser249.

How can I confirm the specificity of the Phospho-RUNX1 (S249) Antibody in my experiments?

To confirm antibody specificity:

• Peptide competition assay: Pre-incubate the antibody with excess phospho-peptide used as immunogen (derived from human RUNX1 around Ser249). This should eliminate specific signal in Western blot or immunostaining.

• Phosphatase treatment: Treat half of your sample with lambda phosphatase before Western blotting. The signal should disappear in treated samples but remain in untreated controls.

• Genetic validation: Use RUNX1 knockout cells or tissues, or cells expressing a RUNX1 S249A mutant (which cannot be phosphorylated at this site) as negative controls.

• Stimulation experiments: Treat cells with agents known to induce RUNX1 S249 phosphorylation (e.g., IL-6 or HIPK2 activators) and demonstrate increased antibody reactivity .

• Correlation with known expression patterns: RUNX1 phospho-S249 should be detected in tissues known to express RUNX1, including thymus, bone marrow, and peripheral blood, but not in brain or heart tissue .

What are common problems encountered with this antibody and how can they be resolved?

Common issues and solutions:

• Weak or no signal in Western blot:

  • Increase primary antibody concentration (try 1:500 instead of 1:2000)

  • Extend primary antibody incubation time to overnight at 4°C

  • Ensure phosphatase inhibitors are included in sample preparation

  • Use fresh antibody; avoid repeated freeze-thaw cycles

  • Try different blocking agents (BSA instead of milk, which contains phosphatases)

• High background:

  • Decrease primary antibody concentration

  • Increase washing steps in duration and number

  • Use freshly prepared buffers

  • Ensure blocking is sufficient (try 5% BSA in TBST)

• Multiple bands or unexpected molecular weight:

  • RUNX1 has multiple isoforms and can undergo various post-translational modifications

  • Expected molecular weight is 49 kDa, but bands at 55 kDa and 70 kDa may also be observed

  • Verify with a total RUNX1 antibody to confirm band pattern

• Inconsistent results between experiments:

  • Standardize lysate preparation protocol

  • Monitor phosphorylation status immediately after sample collection

  • Ensure consistent treatment conditions between experiments

How should I quantify and normalize Phospho-RUNX1 (S249) levels in Western blot experiments?

For accurate quantification:

• Normalization methods:

  • Normalize phospho-RUNX1 (S249) to total RUNX1 protein (run on parallel blots or after stripping and reprobing) to determine the proportion of phosphorylated protein

  • For loading control, use housekeeping proteins like GAPDH, β-actin, or α-tubulin

• Quantification approach:

  • Use densitometry software (ImageJ, Image Lab, etc.) to measure band intensity

  • Calculate the ratio of phospho-RUNX1 to total RUNX1

  • Compare this ratio between experimental conditions

• Statistical analysis:

  • Perform experiments in at least triplicate

  • Use appropriate statistical tests (t-test, ANOVA) to determine significance

  • Report data as "relative intensity of pRunx1 Ser249 (pRunx1 Ser249/Runx1)" as shown in published literature

• Controls for normalization:

  • Include a standard sample across all blots for inter-blot comparison

  • Consider using a standard curve of recombinant phosphorylated protein for absolute quantification

How is RUNX1 S249 phosphorylation regulated and what are its functional implications?

RUNX1 S249 phosphorylation is regulated through several mechanisms:

• Kinase pathways:

  • Homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates RUNX1 at S249, T273, and S276 when RUNX1 is associated with CBFB and DNA

  • MAPK/ERK signaling pathway has been implicated in RUNX1 phosphorylation

  • IL-6 treatment induces phosphorylation in the C-terminus of RUNX1

• Functional implications:

  • Phosphorylation enhances RUNX1 interaction with KAT6A (histone acetyltransferase)

  • Promotes subsequent EP300 (histone acetyltransferase) phosphorylation

  • Affects the stabilization and transcriptional activity of RUNX1

  • May regulate RUNX1's role as a transcription factor in binding to target DNA sequences (5'-TGTGGT-3' or 5'-TGCGGT-3')

• Disease relevance:

  • Altered phosphorylation may contribute to leukemogenesis in conditions involving RUNX1 abnormalities

  • Phosphorylated RUNX1 has been implicated in bone cancer-associated pain mechanisms

What is the relationship between RUNX1 phosphorylation at S249 and hematological disorders?

RUNX1 is a critical transcription factor in hematopoiesis, and its dysregulation is associated with several blood disorders:

• Acute Myeloid Leukemia (AML):

  • RUNX1 (also known as AML1) is frequently involved in chromosomal translocations in AML

  • The t(8;21) translocation results in the AML1-ETO fusion protein, which disrupts normal RUNX1 function

  • Phosphorylation status may affect the function of both wild-type and mutant RUNX1 proteins

• Chronic Myelogenous Leukemia (CML):

  • Chromosomal aberrations involving RUNX1/AML1, such as translocation t(3;21)(q26;q22) with EAP, MSD1, or EVI1 are associated with CML

  • Altered phosphorylation may contribute to disease progression

• Chronic Myelomonocytic Leukemia:

  • Inversion inv(21)(q21;q22) with USP16 involving RUNX1/AML1 is linked to this condition

• Functional hematopoietic changes:

  • In mouse models with RUNX1 mutations affecting post-translational modifications (Runx1KTAMK/KTAMK), changes in peripheral blood cell populations are observed, including reduced CD4 single positive cells

  • These mice show altered hematological parameters including decreased lymphocyte percentages (67.83% vs. 85.27% in wild-type) and increased neutrophil percentages (27.52% vs. 12.06% in wild-type)

Studying RUNX1 S249 phosphorylation may provide insights into disease mechanisms and potential therapeutic targets.

How can the Phospho-RUNX1 (S249) Antibody be used in non-hematopoietic research contexts?

While RUNX1 is predominantly studied in hematopoietic contexts, research has revealed roles in other tissues:

• Neurological research:

  • Recent studies show RUNX1 involvement in sensory neuron function

  • Phosphorylated RUNX1 (S249) levels increase in dorsal root ganglion (DRG) neurons in bone cancer pain models

  • The GDNF-ERK-Runx1 signaling pathway contributes to P2X3R upregulation in nociceptive neurons

  • Researchers can use the antibody to investigate neurological pain mechanisms

• Cancer research beyond hematological malignancies:

  • The antibody can be used to study RUNX1 phosphorylation in solid tumors

  • Potential application in bone metastasis research, where RUNX1 has been implicated in pain mechanisms

• Developmental biology:

  • RUNX1 plays roles in tissue development beyond hematopoiesis

  • The phospho-specific antibody can help track activation states during developmental processes

• Signal transduction studies:

  • Use the antibody to monitor MAPK/ERK pathway activity through RUNX1 phosphorylation

  • Investigate cross-talk between RUNX1 and other signaling pathways

Researchers should validate the antibody in their specific non-hematopoietic system before conducting extensive studies.

How might Phospho-RUNX1 (S249) detection be incorporated into multi-parameter analyses?

Advanced research often requires integrating multiple parameters:

• Flow cytometry applications:

  • Combine with surface markers for hematopoietic lineages

  • Use for intracellular phospho-flow to detect RUNX1 phosphorylation status in specific cell populations

  • Protocol adaptation required: fix cells with paraformaldehyde, permeabilize with methanol or commercial permeabilization buffers

• Multi-omics integration:

  • Correlate RUNX1 phosphorylation data with:

    • Transcriptomics (RNA-seq) to identify genes regulated by phosphorylated RUNX1

    • Proteomics data to understand global phosphorylation networks

    • Chromatin immunoprecipitation (ChIP-seq) to map phospho-RUNX1 binding sites

• Single-cell analysis:

  • Adapt for mass cytometry (CyTOF) with metal-conjugated antibodies

  • Develop imaging mass cytometry protocols to visualize phospho-RUNX1 in tissue contexts

  • Consider single-cell Western blot applications for heterogeneous populations

• Spatial analysis in tissues:

  • Combine with other phospho-specific antibodies in multiplexed immunofluorescence

  • Use with tissue clearing techniques for 3D visualization of phospho-RUNX1 distribution

  • Correlate with in situ hybridization for RUNX1 target genes

These advanced applications require rigorous validation and optimization of the antibody under specific experimental conditions.