RUNX3 Antibody

What is RUNX3 and why is it an important research target?

RUNX3 (also known as CBFA3, AML2, and PEBP2A3) belongs to the Runt domain family of nuclear transcriptional regulators. It plays crucial roles in:

• Regulating gene expression related to cellular differentiation and cell cycle progression

• Functioning as a tumor suppressor, particularly in gastric epithelial cells

• Controlling development in the nervous system (neurogenesis of dorsal root ganglia)

• Mediating T-cell differentiation and immune function

• Acting as a downstream effector in TGF-β signaling pathways

RUNX3 typically forms heterodimeric complexes with core-binding factor beta (CBFβ), which enhances its stability and DNA-binding capability. The complex recognizes the core consensus binding sequence 5'-TGTGGT-3' (or rarely 5'-TGCGGT-3') within regulatory regions of target genes .

What criteria should guide my selection of a RUNX3 antibody for specific applications?

When selecting a RUNX3 antibody, consider these factors based on your experimental needs:

For most applications, monoclonal antibodies like clone A-3 (detects RUNX3 in mouse, rat, and human samples) provide excellent specificity and reproducibility . For certain applications requiring detection of multiple epitopes, polyclonal antibodies may offer advantages.

How do I interpret molecular weight variations when detecting RUNX3 by Western blot?

RUNX3 is typically detected at approximately 44-45 kDa by Western blotting, but researchers may observe bands between 45-55 kDa depending on:

• Post-translational modifications (particularly phosphorylation)

• Different isoforms (human RUNX3 has multiple transcript variants)

• Sample preparation conditions (denaturing vs. native)

The RUNX3 Antibody from Proteintech (27099-1-AP) consistently detects RUNX3 at 45-55 kDa in human, mouse, and rat samples . When using antibodies like clone 2B3, you can expect to observe a band of approximately 45 kDa in Jurkat cell lysates . If you observe unexpected banding patterns, consider using multiple antibodies targeting different epitopes to confirm specificity.

What is the optimal protocol for detecting RUNX3 in fixed tissue samples by immunohistochemistry?

For successful immunohistochemical detection of RUNX3 in fixed tissues:

• Tissue preparation: Use 4% paraformaldehyde fixation followed by paraffin embedding

• Antigen retrieval: Most RUNX3 antibodies require heat-induced epitope retrieval

  • Primary option: Use TE buffer pH 9.0 for optimal results

  • Alternative: Citrate buffer pH 6.0 may be effective for some antibodies

• Antibody selection and dilution:

  • For paraffin sections, use antibodies validated for IHCP (e.g., RUNX3 Antibody A-3)

  • Start with manufacturer's recommended dilution (typically 1:50-1:500)

• Detection system: Use a high-sensitivity detection system (ABC or polymer-based)

• Counterstaining: Hematoxylin works well to visualize nuclear localization of RUNX3

• Controls: Include positive controls (tonsil, spleen, or small intestine tissue)

When scoring RUNX3 expression in tissues, consider using a combined approach that accounts for both percentage of positive cells and staining intensity:

The final score can be calculated as PS × IS to provide a semi-quantitative assessment of RUNX3 expression .

How can I optimize intracellular RUNX3 staining for flow cytometry?

For optimal detection of RUNX3 by flow cytometry in immune cells:

• Cell preparation:

  • Isolate cells (PBMCs or cultured cells) and wash in PBS containing 1% BSA

  • For stimulation experiments, treat cells with PMA (50 ng/mL) to upregulate RUNX3 expression

• Fixation and permeabilization:

  • Fix cells with paraformaldehyde (2-4%)

  • Permeabilize with saponin-based buffer (critical for nuclear antigen access)

• Antibody staining:

  • Use directly conjugated antibodies when available (e.g., PE-conjugated anti-RUNX3, clone R3-5G4)

  • For unconjugated primary antibodies, follow with appropriate fluorochrome-conjugated secondary antibodies

  • Include proper isotype controls (same concentration as the primary antibody)

• Instrument settings:

  • Optimize voltages for the specific fluorochrome

  • Include single-stained controls for compensation

  • Set gates based on isotype control or unstimulated cells

When analyzing data, be aware that RUNX3 expression varies significantly between different immune cell populations, with highest expression in CD8+ T cells and NK cells .

What is the most reliable approach for using RUNX3 antibodies in ChIP assays to identify genomic binding sites?

For effective chromatin immunoprecipitation (ChIP) of RUNX3:

• Cross-linking and chromatin preparation:

  • Cross-link protein-DNA complexes with 1% formaldehyde for 10 minutes

  • Sonicate chromatin to fragments of 200-500 bp

  • Verify fragment size by agarose gel electrophoresis

• Antibody selection:

  • Use ChIP-validated RUNX3 antibodies

  • Consider also using antibodies against CBFβ, which forms heterodimers with RUNX3

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate with RUNX3 antibody overnight at 4°C

  • Include negative controls (IgG) and positive controls (input chromatin)

• PCR primers design:

  • Design primers spanning RUNX3 consensus binding sites (5'-TGTGGT-3')

  • For FOXP3 promoter analysis, target regions containing the three putative RUNX binding sites at positions -333, -287, and -53 relative to the transcription start site

• Data analysis:

  • Analyze enrichment relative to input and IgG controls

  • Consider ChIP-seq for genome-wide binding site identification

This approach has successfully identified RUNX3 binding to the FOXP3 promoter during TGF-β-induced regulatory T cell differentiation .

How can RUNX3 antibodies be used to investigate its role in TGF-β-mediated immune regulation?

RUNX3 plays a critical role in TGF-β signaling and immune regulation, particularly in regulatory T cell (Treg) development. To investigate this relationship:

• Co-immunoprecipitation studies:

  • Use RUNX3 antibodies (e.g., clone A-3) for immunoprecipitation

  • Probe for interaction partners in the TGF-β pathway

  • Identify protein complexes formed during Treg differentiation

• Inducible Treg differentiation models:

  • Culture naive CD4+ T cells with anti-CD2/3/28 antibodies and TGF-β

  • Monitor RUNX3 expression over time using flow cytometry or western blotting

  • Correlate RUNX3 levels with FOXP3 expression

• siRNA knockdown experiments:

  • Transfect naive CD4+ T cells with RUNX3-specific siRNA

  • Assess impact on TGF-β-induced FOXP3 expression

  • Measure functional suppressive capacity of resulting Tregs

Research has shown that TGF-β induces both RUNX1 and RUNX3 expression in CD4+ T cells, which subsequently bind to the FOXP3 promoter at three conserved sites. This binding is essential for optimal FOXP3 expression and regulatory T cell function. Combined knockdown of both RUNX1 and RUNX3 has a more profound effect on reducing FOXP3 expression than targeting either factor alone, suggesting functional redundancy .

What techniques can be used to study RUNX3's role as a tumor suppressor in cancer progression?

To investigate RUNX3's tumor suppressor function:

• Epigenetic analysis of RUNX3 promoter:

  • Analyze hypermethylation of RUNX3 gene's CpG island in cancer samples

  • Compare RUNX3 promoter methylation status between normal and cancer tissues

  • Correlate methylation patterns with RUNX3 expression levels

• Protein degradation and stability studies:

  • Examine post-translational modifications affecting RUNX3 stability

  • Study ubiquitination pathways targeting RUNX3

  • Investigate RUNX3 interaction with E3 ligases like β-TrCP

• Functional interaction with oncogenic pathways:

  • Study RUNX3's direct interaction with oncogenes like GLI1

  • Analyze how RUNX3 affects GLI1 ubiquitination and degradation

  • Assess impact on downstream signaling pathways

RUNX3 has been shown to suppress metastasis and stemness in colorectal cancer by inhibiting GLI1, a key component of the Hedgehog signaling pathway. RUNX3 directly interacts with GLI1, promoting its ubiquitination through the E3 ligase β-TrCP. This regulatory mechanism limits Hedgehog signaling during tumor development. In colorectal cancer tissues, RUNX3 and GLI1 expression are inversely correlated, and loss of RUNX3 combined with increased GLI1 predicts poor survival and increased metastasis .

How can I design experiments to investigate the cross-talk between RUNX3 and other transcription factors?

To study RUNX3's interactions with other transcription factors:

• Co-immunoprecipitation followed by mass spectrometry:

  • Use validated RUNX3 antibodies for pull-down experiments

  • Identify novel interaction partners

  • Confirm interactions with co-IP in reverse direction

• Dual luciferase reporter assays:

  • Create reporter constructs with promoters containing RUNX binding sites

  • Co-transfect with RUNX3 and potential partner transcription factors

  • Measure cooperative or antagonistic effects on transcriptional activity

  • Include mutated binding site controls

• Sequential ChIP (Re-ChIP):

  • Perform initial ChIP with RUNX3 antibody

  • Re-immunoprecipitate with antibodies against potential partners

  • Analyze co-occupancy at specific genomic loci

• Proximity ligation assay:

  • Detect protein-protein interactions in situ

  • Use antibodies against RUNX3 and suspected interaction partners

  • Visualize interactions as fluorescent dots by microscopy

For example, research has demonstrated that RUNX3 cooperates with ZFHX3 to upregulate CDKN1A promoter activity following TGF-β stimulation . Additionally, RUNX3 and FOXP3 colocalization has been observed in human tonsil regulatory T cells, suggesting functional interaction in these immunosuppressive cells .

What are common pitfalls when working with RUNX3 antibodies and how can I address them?

For Western blot applications, RUNX3 antibody (A-3) has been validated to detect RUNX3 protein from mouse, rat, and human origin , while clone 2B3 reliably detects a band of approximately 45 kDa in Jurkat cell lysates .

How can I validate the specificity of my RUNX3 antibody?

To ensure antibody specificity:

• Positive and negative controls:

  • Use cell lines with known RUNX3 expression (positive: Jurkat cells; negative: cell lines with RUNX3 knockout)

  • Compare staining patterns in tissues with known expression (spleen, tonsil, small intestine)

• Knockdown/knockout validation:

  • Compare antibody reactivity between wild-type and RUNX3 knockout cell lines

  • Use RUNX3 siRNA-treated cells as specificity controls

• Peptide competition assays:

  • Pre-incubate antibody with excess immunizing peptide

  • Observe elimination of specific signal

• Multiple antibody approach:

  • Use antibodies recognizing different epitopes

  • Compare detection patterns across techniques

Ab135248 (clone 2B3) has been validated in RUNX3 knockout HAP1 cells, showing loss of the expected 44 kDa band in knockout cells while preserving non-specific cross-reactive bands, providing clear evidence of specificity .

How can I determine the optimal RUNX3 antibody concentration for my specific experimental conditions?

For optimal antibody titration:

• Western blotting:

  • Test dilution series (typically 1:500-1:2000)

  • Assess signal-to-noise ratio

  • Select concentration that provides clear specific bands with minimal background

• Immunohistochemistry:

  • Prepare a dilution series (typically 1:50-1:500)

  • Test on known positive tissue sections

  • Select concentration that gives specific staining with minimal background

  • Consider different antigen retrieval methods for each dilution

• Flow cytometry:

  • Titrate antibody using known positive cells

  • Compare signal separation between positive and negative populations

  • Calculate staining index (median positive - median negative)/2 × SD negative

  • Select concentration with highest staining index

• Documentation:

  • Record lot number, dilution, and conditions

  • Include positive controls when switching to a new lot

  • Consider preparing working stock solutions to maintain consistency

The optimal antibody concentration may vary between applications and sample types, so separate titration experiments should be performed for each new experimental system.

How can RUNX3 antibodies be used to investigate its role in T cell lineage commitment and function?

RUNX3 plays critical roles in T cell development and function. To investigate these roles:

• Developmental studies:

  • Use flow cytometry with RUNX3 antibodies to track expression during T cell development

  • Analyze RUNX3 expression in thymocyte subsets (DN, DP, CD4SP, CD8SP)

  • Correlate RUNX3 levels with commitment to CD8+ lineage

• Functional studies in differentiated T cells:

  • Examine RUNX3 expression in different T cell subsets (CD8+, CD4+, Tregs)

  • Analyze correlation between RUNX3 levels and cytotoxic function in CD8+ T cells

  • Study RUNX3 expression in CD4+CD8αα intraepithelial lymphocytes

• Regulatory T cell investigations:

  • Compare RUNX3 expression in human CD4+CD25highCD127- Tregs versus conventional CD4+ T cells

  • Analyze co-expression with FOXP3 in regulatory populations

  • Assess impact of RUNX3 knockdown on suppressive function

Research has demonstrated that RUNX3 is highly expressed in human CD4+CD25highCD127- regulatory T cells compared to conventional CD4+ T cells, with significant co-expression of FOXP3 and TGF-β mRNA. RUNX3 and FOXP3 have been shown to colocalize in human tonsil regulatory T cells, suggesting functional interaction in these immunosuppressive cells .

What are the latest techniques for studying RUNX3 binding site occupancy genome-wide?

For genome-wide analysis of RUNX3 binding:

• ChIP-sequencing:

  • Use highly specific RUNX3 antibodies for immunoprecipitation

  • Prepare sequencing libraries from precipitated DNA

  • Identify genome-wide binding patterns and motifs

  • Cross-reference with gene expression data

• CUT&RUN or CUT&TAG:

  • These techniques offer improved signal-to-noise ratio over traditional ChIP

  • Require fewer cells and less antibody

  • Provide higher resolution binding site identification

• ChIP-SICAP (Selective Isolation of Chromatin-Associated Proteins):

  • Combines ChIP with proximity labeling

  • Identifies proteins associated with RUNX3 at specific genomic loci

• HiChIP:

  • Combines chromatin immunoprecipitation with Hi-C

  • Maps long-range interactions mediated by RUNX3

  • Reveals 3D genome organization at RUNX3 binding sites

When designing these experiments, focus on RUNX3's known consensus binding sequence (5'-TGTGGT-3') and include analysis of RUNX3's binding partner CBFβ to fully understand the functional consequences of binding site occupancy.

How can I investigate RUNX3's role in drug response and potential therapeutic applications?

To study RUNX3 in therapeutic contexts:

• Drug-induced RUNX3 expression changes:

  • Treat cells with candidate compounds (e.g., epigenetic modulators)

  • Monitor RUNX3 expression changes by qPCR, Western blot, and immunofluorescence

  • Correlate RUNX3 induction with phenotypic changes

• RUNX3 as a biomarker for treatment response:

  • Analyze RUNX3 expression or promoter methylation in patient samples

  • Correlate with treatment outcomes

  • Develop immunohistochemical protocols with standardized scoring

• Therapeutic targeting of RUNX3 pathways:

  • Identify downstream effectors using RUNX3 overexpression or knockdown

  • Target these pathways with small molecules

  • Monitor pathway activity using RUNX3 antibodies in combination with phospho-specific antibodies

• Combination therapy approaches:

  • Test combinations of RUNX3-inducing agents with standard therapies

  • Analyze synergistic effects on cancer cell growth and invasion

  • Use RUNX3 antibodies to confirm mechanism of action

Research has shown that RUNX3 inactivation through hypermethylation is a common event in gastric cancer, making it a potential biomarker for diagnosis and risk assessment. Detection of hypermethylation in RUNX3 gene's CpG island can provide valuable insights into gastric cancer pathology and potentially guide treatment decisions .