RUNX3 Antibody, Biotin conjugated

What is RUNX3 and why is it significant in cancer research?

RUNX3 functions as a tumor suppressor, with its inactivation implicated in various cancer types. The protein suppresses gastric epithelial cell growth through p21 induction, cooperating with SMADs to synergistically activate the p21 promoter . Notably, the RUNX3-R122C mutation identified in gastric cancer patients abolishes this ability to activate the p21 promoter or cooperate with SMADs . RUNX3 also inhibits invasion and migration in esophageal squamous cell carcinoma (ESCC) by reversing epithelial-mesenchymal transition (EMT) through TGF-β/Smad signaling . Detection of RUNX3 using specific antibodies is therefore essential for understanding its biological roles and potential clinical applications.

What experimental applications are appropriate for biotin-conjugated RUNX3 antibodies?

Biotin-conjugated RUNX3 antibodies are versatile tools applicable across multiple experimental techniques:

• Immunohistochemistry (IHC): Used to detect RUNX3 expression in tissue samples, as demonstrated in studies examining RUNX3 expression in ESCC tissues

• Western blotting: For protein expression analysis in cell and tissue lysates

• Chromatin immunoprecipitation (ChIP): To study RUNX3 interaction with target gene promoters like p21 and Suv39H1

• Immunoprecipitation (IP): For investigating protein-protein interactions, such as those between RUNX3 and SAV1 or MST2

• Flow cytometry: For quantitative analysis of RUNX3 expression in cell populations

• Protein-protein interaction studies: Especially useful in avidin-biotin complex formation for enhanced detection sensitivity

How does RUNX3 protein expression vary across different tissue types?

RUNX3 expression patterns vary significantly across tissues, which is important to consider when planning experiments:

• Gastric mucosa: Areas in mouse and human gastric epithelium show coordinated expression of RUNX3 and p21

• Esophageal tissue: RUNX3 expression is significantly reduced in ESCC tissue compared to adjacent normal tissue (0.50±0.20 vs. 0.83±0.16; P<0.001)

• Cancer tissues: Often show decreased nuclear RUNX3 expression compared to adjacent normal tissues, with statistical analysis revealing associations between decreased RUNX3 expression and disease progression factors such as T status (P=0.027) and lymph node metastasis (P=0.017) in ESCC patients

How should researchers select controls for RUNX3 antibody experiments?

Appropriate controls are critical for RUNX3 antibody experiments:

• Positive controls: Use tissues or cell lines with known RUNX3 expression (SNU16 cells express endogenous RUNX3)

• Negative controls: Replace primary antibodies with phosphate-buffered saline (PBS) to rule out nonspecific binding

• Experimental controls: When studying RUNX3 overexpression, compare with cells stably transfected with empty vector

• Isotype controls: Use appropriate isotype-matched antibodies to assess background

• Genetic controls: Consider RUNX3-knockdown or knockout models as definitive negative controls

How can researchers optimize biotin-conjugated RUNX3 antibodies for chromatin immunoprecipitation (ChIP)?

Optimizing RUNX3 antibodies for ChIP requires careful consideration of several parameters:

• Cell preparation: For each ChIP experiment, use approximately 1×10^7 cells fixed for 15 minutes at room temperature with 1% formaldehyde-containing medium

• Chromatin preparation: Isolate nuclei and sonicate chromatin to an average size of 220 bp in shearing buffer with protease inhibitor cocktail

• Immunoprecipitation: Incubate sonicated chromatin with anti-RUNX3 antibodies (40 μg) overnight at 4°C

• Bead selection: Use protein A-coated magnetic beads (120 μl per ChIP reaction) with 2-hour incubation at 4°C

• Elution protocol: Incubate beads in 400 μl elution buffer at 65°C for 4 hours to elute immunoprecipitated materials

• Validation: Perform ChIP-seq assays using chromatin and input controls from three different cell cultures for reproducibility

What methods can detect RUNX3-protein interactions using biotin-conjugated antibodies?

Several approaches are effective for studying RUNX3 protein interactions:

• Co-immunoprecipitation (Co-IP): This method has successfully demonstrated interactions between RUNX3 and partners such as SAV1 and MST2 . Co-IP assays have shown that RUNX3 antibody can enrich interacting proteins like MEX3C .

• In vitro translation systems: The TnT Quick Coupled Translation System can generate HA-tagged RUNX3 proteins for interaction studies .

• Pull-down assays: RNA pull-down assays using RUNX3 antibody-conjugated probes have demonstrated enrichment of interacting proteins like Suv39H1 .

• Mutant protein studies: Creating RUNX3 mutants through site-directed mutagenesis enables mapping of specific interaction domains .

• Luciferase reporter assays: Used to verify RUNX3 binding to promoter regions of target genes like Suv39H1 .

How do post-translational modifications affect RUNX3 detection and function?

Post-translational modifications significantly impact RUNX3 detection and biological activity:

• Ubiquitination: MEX3C induces ubiquitylation and subsequent degradation of RUNX3 . This modification affects protein stability and detection in proteasome-dependent manner, as shown by reversing MEX3C's effect on RUNX3 protein levels using the proteasome inhibitor MG132 .

• Phosphorylation: MST2 pathway activation affects RUNX3 activity and protein interactions, particularly with SAV1 .

• Nuclear localization: RUNX3 functions as a transcription factor in the nucleus, and detection methods must consider its subcellular localization for accurate interpretation .

• Epitope masking: Some modifications may affect antibody binding to specific epitopes, requiring careful antibody selection based on the target epitope region.

What approaches help resolve contradictory RUNX3 expression data between studies?

When encountering discrepancies in RUNX3 expression data, consider these methodological approaches:

• Antibody validation: Verify antibody specificity through multiple methods including western blot, immunoprecipitation, and genetic controls.

• Multiple detection methods: Compare results from different techniques (IHC, western blot, qRT-PCR) as demonstrated in studies that used both protein and mRNA expression analysis .

• Standardized protocols: Use consistent sample preparation, fixation, and antigen retrieval methods across experiments.

• Statistical validation: Apply appropriate statistical tests for data analysis, such as paired Student's t-test for comparing RUNX3 in paired tumor tissues and adjacent normal tissues, and unpaired tests for other comparisons .

• Biological context: Consider cell-type specific regulation and subcellular localization differences that might explain apparent contradictions.

How can biotin-conjugated RUNX3 antibodies help investigate RUNX3 degradation pathways?

RUNX3 degradation studies can be enhanced using biotin-conjugated antibodies:

• Ubiquitination assays: Vectors expressing tagged proteins (e.g., Myc-tagged MEX3C, HA-tagged ubiquitin, and Flag-tagged RUNX3) can be transfected into cells to study ubiquitination patterns .

• Proteasome inhibition studies: Compare RUNX3 levels with and without proteasome inhibitors like MG132 to assess degradation mechanisms .

• Pulse-chase experiments: Track RUNX3 stability over time under different conditions.

• Co-localization studies: Examine RUNX3 interaction with degradation machinery components.

• Mutant analysis: Compare degradation rates between wild-type RUNX3 and mutants like RUNX3-R122C .

What are optimal fixation and staining protocols for RUNX3 immunohistochemistry?

For successful RUNX3 immunohistochemistry:

• Fixation: Formalin fixation is commonly used in RUNX3 studies

• Antibody dilution: Anti-RUNX3 antibodies are typically used at 1:100 dilution for IHC applications

• Staining method: The streptavidin-peroxidase (SP) method works well with RUNX3 antibodies

• Antigen retrieval: May be necessary depending on fixation method and tissue type

• Controls: Include primary antibody replacement with PBS as negative control

• Visualization: DAB (3,3'-diaminobenzidine) substrate is commonly used for visualization of biotinylated antibody binding

• Signal assessment: Nuclear staining is typically expected for RUNX3 in normal tissues

What technical considerations are important when using biotin-conjugated RUNX3 antibodies in western blotting?

For optimal western blotting with biotin-conjugated RUNX3 antibodies:

• Sample preparation: Proper protein extraction techniques specific to the cellular compartment of interest (nuclear vs. cytoplasmic)

• Electrophoresis conditions: SDS-polyacrylamide gel electrophoresis with appropriate percentage based on RUNX3 molecular weight

• Transfer methods: Transfer to polyvinylidene difluoride membrane for optimal protein binding

• Blocking: Use appropriate blocking solutions to prevent non-specific binding

• Detection systems: Use fluorescent imaging systems like FLA-5000 for radioactive imaging or LAS-3000 for protein quantification

• Controls: Include appropriate loading controls such as GAPDH

• Quantification: Express results as RUNX3/GAPDH ratio for accurate relative quantification

How can researchers troubleshoot weak or non-specific signals with biotin-conjugated RUNX3 antibodies?

When encountering signal issues with biotin-conjugated RUNX3 antibodies:

• Endogenous biotin: Block endogenous biotin using commercial avidin/biotin blocking kits

• Antibody concentration: Titrate antibody concentration to optimize signal-to-noise ratio

• Incubation conditions: Adjust antibody incubation time and temperature

• Washing stringency: Increase washing steps to reduce background

• Detection sensitivity: Consider signal amplification methods for weak signals

• Sample quality: Ensure proper sample preparation and storage

• Antigen retrieval: Optimize antigen retrieval methods for fixed tissues

• Blocking optimization: Test different blocking reagents (BSA, normal serum, commercial blockers)

What statistical approaches are appropriate for analyzing RUNX3 expression data?

Statistical analysis of RUNX3 expression data should be rigorous:

• For comparing RUNX3 in paired tumor and normal tissues: Use paired Student's t-test

• For comparing different groups: Use unpaired Student's t-tests

• For multiple group comparisons: Apply one-way analysis of variance (ANOVA) followed by Tukey's post hoc test

• For correlations: Use Pearson's correlation analysis to evaluate relationships between RUNX3 and other markers (e.g., EMT-related markers)

• For associating RUNX3 expression with clinicopathological variables: Apply χ² test

• Significance threshold: Define statistical significance as P<0.05

• Data presentation: Express quantitative data as mean ± standard deviation (SD)

What are the key validation steps for confirming RUNX3 antibody specificity?

Comprehensive validation of RUNX3 antibodies should include:

• Expression confirmation: Verify exogenous RUNX3 expression by Northern or Western blotting

• Mutation testing: Test antibody recognition of RUNX3 mutants (e.g., RUNX3-R122C)

• Knockout/knockdown controls: Compare staining in RUNX3-depleted samples

• Peptide competition: Pre-absorb antibody with immunizing peptide

• Multiple antibodies: Compare results with antibodies targeting different RUNX3 epitopes

• Cross-reactivity assessment: Test against related proteins (RUNX1, RUNX2)

• Correlation with mRNA: Compare antibody-based detection with mRNA expression analysis

How does RUNX3 contribute to cell cycle regulation and apoptosis?

RUNX3's role in cell cycle control and apoptosis involves several mechanisms:

• p21 induction: RUNX3 suppresses gastric epithelial cell growth by inducing p21, a cell cycle inhibitor. This effect is synergistically enhanced through cooperation with SMADs .

• SMAD pathway interaction: RUNX3 cooperates with SMADs to activate the p21 promoter, establishing a link between RUNX3 and TGF-β signaling .

• Mutation effects: The RUNX3-R122C mutation identified in gastric cancer patients abolishes the ability to activate the p21 promoter or cooperate with SMADs, highlighting the importance of this mechanism in tumor suppression .

• MST pathway involvement: RUNX3 forms a complex with SAV1 in an MST2-dependent manner, linking it to the Hippo tumor suppressor pathway .

• Oncogenic Ras response: RUNX3 is stabilized by Ras activation through the p14 ARF-MDM2 signaling pathway and plays an essential role in oncogenic Ras-induced apoptosis .

What is known about RUNX3's role in epithelial-mesenchymal transition (EMT)?

RUNX3's impact on EMT is significant for understanding cancer progression:

• EMT marker correlation: RUNX3 expression in ESCC tissues negatively correlates with N-cadherin (r=−0.429; P<0.01) and Snail (r=−0.364; P<0.01) expression, while positively correlating with E-cadherin expression (r=0.580; P<0.01) .

• Invasion and migration inhibition: RUNX3 overexpression significantly inhibits Eca109 and EC9706 cell invasion, migration, MMP-9 expression, and EMT .

• TGF-β/Smad signaling: RUNX3 overexpression markedly inhibits the phosphorylation of Smad2/3, and RUNX3-overexpressing cells display less sensitivity to TGF-β1-induced EMT than control cells .

• Clinical correlations: Statistical analysis reveals associations between decreased RUNX3 expression and T status (P=0.027) and lymph node metastasis (P=0.017) in ESCC patients .

What emerging techniques are advancing RUNX3 research using antibody-based approaches?

Novel methodological approaches in RUNX3 research include:

• ChIP-seq analysis: Advanced chromatin immunoprecipitation followed by sequencing identifies genome-wide RUNX3 binding sites .

• Protein interaction studies: In vitro translation systems combined with immunoprecipitation reveal novel RUNX3 binding partners .

• Site-directed mutagenesis: Generating RUNX3 mutants to map functional domains and interaction sites .

• Ubiquitination assays: Sophisticated systems to study RUNX3 degradation pathways and regulators like MEX3C .

• Reporter gene assays: Luciferase reporter systems with mutational analysis to map RUNX3 binding sites in target gene promoters like Suv39H1 .

• RNA-protein interaction analysis: RNA pull-down assays to investigate RUNX3's interactions with RNA and RNA-binding proteins .

How can biotin-conjugated RUNX3 antibodies contribute to cancer biomarker research?

Applications of RUNX3 antibodies in cancer biomarker development:

• Expression profiling: IHC analysis of RUNX3 in cancer tissues compared to adjacent normal tissues provides prognostic information .

• Correlation with clinical parameters: Statistical analyses relating RUNX3 expression to clinicopathological variables help establish its biomarker potential .

• Multi-marker panels: Combining RUNX3 detection with EMT markers (E-cadherin, N-cadherin, Snail) improves prognostic value .

• Subcellular localization assessment: Nuclear versus cytoplasmic RUNX3 localization provides crucial diagnostic information .

• Epigenetic regulation monitoring: Evaluating RUNX3 methylation status alongside protein expression can be a powerful combined biomarker .

• Protein modification patterns: Detecting specific RUNX3 post-translational modifications like ubiquitination may serve as novel biomarkers .

What considerations are important when using multiple antibodies targeting different RUNX3 epitopes?

When employing multiple RUNX3 antibodies:

• Epitope mapping: Different antibodies recognize distinct RUNX3 domains (N-terminal, Runt domain, C-terminal) .

• Mutation effects: Some RUNX3 mutations (like R122C) may affect epitope recognition by specific antibodies .

• Post-translational modifications: Modifications near specific epitopes may interfere with antibody binding.

• Validation strategies: Confirm concordance between antibodies targeting different epitopes.

• Application optimization: Each antibody may require different optimization for specific applications (Western blot, IHC, ChIP) .

• Interpretation consistency: Standardize scoring and interpretation methods when using different antibodies.

What future directions are emerging in RUNX3 antibody-based research?

The field of RUNX3 research continues to evolve with several promising directions:

• Integration with multi-omics approaches: Combining antibody-based protein detection with genomics, transcriptomics, and epigenomics data.

• Development of therapeutic antibodies: Exploring RUNX3-targeted therapies based on understanding its regulatory mechanisms.

• Single-cell applications: Adapting RUNX3 antibody techniques for single-cell protein analysis.

• In vivo imaging: Developing biotin-conjugated RUNX3 antibodies for in vivo molecular imaging applications.

• Liquid biopsy applications: Detecting RUNX3 or its modified forms in circulating tumor cells or exosomes.

• Advanced multiplex systems: Incorporating RUNX3 antibodies into high-dimensional protein analysis platforms.