RUNX1 Antibody, FITC conjugated

What is RUNX1 and why is it a significant research target?

RUNX1 (also known as AML1, CBF-alpha-2, and CBFA2) functions as a critical hematopoietic transcription factor expressed from two different promoters: the proximal P2 promoter produces RUNX1B while the distal P1 promoter yields RUNX1C . These isoforms differ structurally, with RUNX1C containing 32 additional amino acids at the N-terminus compared to RUNX1B . The significance of RUNX1 stems from its fundamental role in hematopoietic development and its involvement in various pathological conditions, particularly leukemias. Mutations in RUNX1 are associated with Familial Platelet Disorder with predisposition to Acute Myeloid Leukemia (FPDMM), making RUNX1 a crucial research target for understanding hematological malignancies and potential therapeutic interventions .

What are the key considerations when selecting a FITC-conjugated RUNX1 antibody?

When selecting a FITC-conjugated RUNX1 antibody, researchers should consider several critical factors to ensure experimental success. First, determine the specific isoform recognition requirements – whether you need an antibody that recognizes all RUNX1 isoforms or one that specifically targets RUNX1B or RUNX1C. Research has demonstrated that these isoforms have differential regulatory effects on gene expression . Second, verify the host species compatibility to avoid cross-reactivity issues; validated RUNX1 antibodies typically confirm reactivity with human, bovine, and monkey samples . Third, check the validated applications – FITC-conjugated RUNX1 antibodies are commonly validated for Western blotting and ELISA, with specific recommended dilutions (typically 1:500 for Western blot and 1:10,000 for ELISA) . Finally, consider the emission/excitation properties of the FITC conjugate (excitation: 490nm, emission: 525nm) to ensure compatibility with your detection equipment and experimental design .

How do RUNX1B and RUNX1C isoforms differentially regulate gene expression?

RUNX1B and RUNX1C isoforms exhibit opposing regulatory effects on both RUNX1 expression itself and downstream target genes. In chromatin immunoprecipitation and luciferase promoter assays, both isoforms bind to P1 and P2 promoters, but with different functional outcomes . In cells lacking endogenous RUNX1, RUNX1B decreases while RUNX1C increases P1 and P2 promoter activities . When overexpressed in megakaryocytic HEL cells, RUNX1B inhibits RUNX1C and RUNX1A expression, whereas RUNX1C upregulates both RUNX1B and RUNX1A expression . This autoregulation has significant implications for hematopoietic development and disease progression.

Beyond autoregulation, these isoforms differentially affect downstream target genes. In HEL cells, RUNX1B and RUNX1C differently regulate target genes including MYL9, F13A1, PCTP, and PDE5A . In platelet studies, RUNX1B transcript levels correlate positively with F13A1, PCTP, PDE5A, and RAB1B expression, but negatively with MYL9 expression . These differential regulatory patterns have clinical relevance, as higher expression of RUNX1 targets F13A1 and RAB31 is associated with acute cardiovascular events in patients with cardiovascular disease .

What methodological approaches are recommended for RUNX1 antibody validation?

Thorough validation of RUNX1 antibodies requires a multi-faceted approach to ensure specificity and reliability. Begin with negative control testing in cell lines lacking endogenous RUNX1 expression (such as HeLa cells) to confirm absence of non-specific binding . Follow with positive control experiments using cells known to express RUNX1 (like HEL cells) where RUNX1 antibodies should detect bands corresponding to RUNX1B (48 kDa) and RUNX1C (52 kDa) . Develop and validate isoform-specific antibodies by generating antibodies targeting unique epitopes, such as the 16 amino acid N-terminus of RUNX1C, and confirm specificity through overexpression experiments .

For functional validation, employ chromatin immunoprecipitation (ChIP) assays to verify binding specificity and locations. Studies have demonstrated that RUNX1 antibodies can enrich regions encompassing RUNX1 consensus binding sites in both P1 and P2 promoters . Additionally, complement antibody-based approaches with genetic manipulations, such as overexpression or knockdown of specific RUNX1 isoforms, to confirm observed effects. Researchers have validated RUNX1 functionality by showing that RUNX1 knockdown attenuates expression of IL-6 and IL-1β in macrophages .

How can FITC-conjugated RUNX1 antibodies be employed in studies of inflammation and immune response?

FITC-conjugated RUNX1 antibodies provide valuable tools for investigating RUNX1's role in inflammation and immune response pathways. Research has revealed that RUNX1 interacts with the NF-κB subunit p50, forming a complex that enhances NF-κB luciferase activity and promotes inflammatory cytokine production in response to lipopolysaccharide (LPS) stimulation . When implementing these antibodies in inflammation studies, researchers should employ flow cytometry to track RUNX1 expression changes in specific immune cell populations during inflammatory responses.

For co-localization studies, combine FITC-conjugated RUNX1 antibodies with antibodies against inflammatory pathway components (labeled with spectrally distinct fluorophores) to visualize protein interactions within cellular compartments during inflammation. RUNX1 silencing experiments have demonstrated that RUNX1 positively regulates IL-1β and IL-6 production in response to LPS stimulation, though TNF-α levels remain unaffected . Researchers can utilize FITC-conjugated RUNX1 antibodies in ChIP-seq experiments to identify genome-wide binding patterns during inflammatory responses, particularly focusing on cytokine gene promoters and enhancers. Additionally, these antibodies can be employed in proximity ligation assays to confirm direct interactions between RUNX1 and NF-κB pathway components in situ, providing spatial resolution to molecular interactions underlying inflammatory responses .

What are the technical considerations for using FITC-conjugated RUNX1 antibodies in flow cytometry?

When implementing FITC-conjugated RUNX1 antibodies in flow cytometry, researchers must address several technical challenges to obtain reliable results. RUNX1 is primarily a nuclear transcription factor, necessitating effective permeabilization protocols to allow antibody access to nuclear compartments. Test multiple permeabilization reagents (such as Triton X-100, saponin, or commercial nuclear permeabilization kits) to determine optimal conditions that maintain cellular integrity while enabling antibody penetration.

FITC has relatively lower brightness and photostability compared to newer fluorophores, which may impact detection sensitivity, particularly with low abundance transcription factors like RUNX1. Consider signal amplification techniques or newer generation FITC derivatives if signal strength is insufficient. To address spectral overlap challenges, especially in multi-parameter experiments, perform thorough compensation controls when FITC is used alongside PE or other fluorophores with spectral proximity. Include appropriate isotype controls matched to the FITC-conjugated RUNX1 antibody concentration to accurately identify positive populations and minimize false positive results.

Fixation timing is critical for nuclear transcription factors like RUNX1, as expression and localization may change rapidly following stimulation. Establish a time-course of fixation points to capture dynamic changes in RUNX1 expression or phosphorylation status. Consider differential expression between RUNX1 isoforms by using antibodies that can distinguish between RUNX1B and RUNX1C, as these have been shown to have distinct regulatory functions in hematopoietic cells .

How can researchers investigate the role of RUNX1 in leukemia cell proliferation?

Investigating RUNX1's role in leukemia cell proliferation requires a comprehensive approach combining antibody-based detection with functional studies. Researchers have demonstrated that RUNX1 upregulates CENPE to promote leukemic cell proliferation, with RUNX1 knockdown significantly reducing cell numbers in THP-1 leukemia cells compared to CD34+ cells . When designing such experiments, establish baseline RUNX1 expression levels in your leukemia model using FITC-conjugated RUNX1 antibodies and flow cytometry or immunofluorescence microscopy.

Implement genetic manipulation strategies through RUNX1 overexpression and knockdown studies to assess direct causality. In published research, RUNX1 overexpression increased cell numbers in both THP-1 and CD34+ cells, with a more pronounced effect in leukemia cells (2-fold increase) compared to normal hematopoietic cells . RUNX1 knockdown produced an 8-fold decrease in THP-1 cells compared to only a 1.5-fold decrease in CD34+ cells, highlighting the differential dependence on RUNX1 in leukemic versus normal cells .

Use FITC-conjugated RUNX1 antibodies in combination with proliferation markers like Ki67 to directly correlate RUNX1 expression with proliferative capacity at the single-cell level. Perform ChIP-seq experiments with RUNX1 antibodies to identify direct transcriptional targets that mediate proliferation effects, such as CENPE and other cell cycle regulators. Additionally, conduct pathway analysis by combining RUNX1 antibody studies with inhibitors of key signaling pathways to determine the molecular mechanisms connecting RUNX1 to proliferation regulation.

What methodology is recommended for studying RUNX1 isoform-specific functions?

Developing a robust methodology for studying RUNX1 isoform-specific functions requires precise tools and carefully designed experiments. Generate or obtain isoform-specific antibodies that selectively recognize unique regions, such as the 32 additional amino acids at the N-terminus of RUNX1C that distinguish it from RUNX1B . Validate these antibodies in systems with controlled expression of individual isoforms, as demonstrated in research where RUNX1C-specific antibodies detected RUNX1C but not RUNX1B in transfected HeLa cells .

Design isoform-specific knockdown strategies using siRNAs or shRNAs targeting unique regions of each transcript. Researchers have successfully employed this approach to demonstrate that RUNX1 isoforms differentially regulate downstream target genes . For overexpression studies, utilize expression vectors containing isoform-specific cDNAs to examine gain-of-function effects. Studies have shown that RUNX1B and RUNX1C have opposing effects on P1 and P2 promoter activities when expressed in HeLa cells lacking endogenous RUNX1 .

Employ isoform-specific ChIP-seq to map genome-wide binding patterns of each isoform, revealing unique and shared target genes. Research has identified that both RUNX1B and RUNX1C bind to regions encompassing RUNX1 consensus sites in P1 and P2 promoters, but with different functional outcomes . Conduct RNA-seq following isoform-specific manipulation to identify comprehensive transcriptional networks regulated by each isoform. Studies have revealed that RUNX1B correlates positively with F13A1, PCTP, PDE5A, and RAB1B expression, while correlating negatively with MYL9 expression in platelets .

What controls should be included when using RUNX1 antibodies in immunofluorescence applications?

Robust experimental design for RUNX1 antibody immunofluorescence requires comprehensive controls to ensure reliable and interpretable results. Include positive control samples from cell types with confirmed RUNX1 expression, such as HEL cells, where RUNX1 antibodies detect bands corresponding to RUNX1B (48 kDa) and RUNX1C (52 kDa) . Incorporate negative control samples from cell types lacking RUNX1 expression, such as HeLa cells, to confirm antibody specificity and establish background signal levels .

Implement technical controls including primary antibody omission controls to assess secondary antibody non-specific binding, isotype controls matched to antibody concentration to evaluate non-specific binding of the primary antibody, and blocking peptide controls using the immunizing peptide to confirm binding specificity. For RUNX1 antibodies, using synthetic peptides from the amino acid region 1-80 of human RUNX1 protein can effectively demonstrate specificity .

Validation controls should include genetic manipulation approaches such as RUNX1 knockdown or knockout samples to confirm signal reduction correlates with reduced protein expression. This has been effectively demonstrated in studies where RUNX1 knockdown significantly reduced cell numbers in THP-1 cells . Include subcellular localization controls targeting known nuclear proteins (for co-localization with RUNX1) to confirm proper fixation and permeabilization procedures. Additionally, employ cross-validation methods using multiple detection techniques (Western blot, qPCR) to confirm expression patterns observed in immunofluorescence.

How can researchers address signal intensity variations when using FITC-conjugated RUNX1 antibodies?

Signal intensity variations present common challenges when using FITC-conjugated antibodies for RUNX1 detection. To address these issues, implement consistent fixation and permeabilization protocols, as variations in these procedures significantly affect nuclear antigen accessibility. Test multiple fixatives (paraformaldehyde, methanol, acetone) at different concentrations and exposure times to determine optimal conditions for RUNX1 detection while preserving cellular morphology.

Titrate antibody concentrations to determine the optimal working dilution for your specific application and cell type. For Western blot applications, a 1:500 dilution is typically recommended for RUNX1 antibodies, while ELISA applications may require more dilute preparations (1:10,000) . Implement signal amplification strategies such as tyramide signal amplification for low-abundance transcription factors like RUNX1, especially when detecting minor isoforms.

Address photobleaching concerns by using anti-fade mounting media for fixed samples and minimizing exposure times during imaging. Consider alternative fluorophores with greater photostability for longitudinal imaging experiments. Standardize image acquisition parameters including exposure time, gain, and laser power across experimental and control samples to ensure comparable signal intensity measurements. Additionally, employ quantitative analysis tools with background subtraction and normalization to internal reference proteins to accurately compare RUNX1 expression levels between experimental conditions.

What strategies can be employed to simultaneously detect RUNX1 and its binding partners?

Simultaneous detection of RUNX1 and its interaction partners requires sophisticated experimental approaches combining multiple labeling techniques. Implement dual immunofluorescence labeling using FITC-conjugated RUNX1 antibodies paired with spectrally distinct fluorophore-conjugated antibodies against known binding partners, such as p50 (NF-κB pathway) . When designing these experiments, carefully select fluorophore combinations to minimize spectral overlap and optimize detection sensitivity.

Utilize proximity ligation assays (PLA) to visualize and quantify direct protein-protein interactions between RUNX1 and its binding partners in situ. This technique can detect interactions within 40nm distance, providing strong evidence for functional protein complexes. For biochemical validation, perform co-immunoprecipitation using RUNX1 antibodies followed by Western blotting for binding partners. Research has demonstrated that RUNX1 interacts with the NF-κB subunit p50, and coexpression of RUNX1 with p50 enhances NF-κB luciferase activity .

Design FRET (Förster Resonance Energy Transfer) experiments using FITC-conjugated RUNX1 antibodies paired with acceptor fluorophore-conjugated antibodies against interaction partners to demonstrate physical proximity in living cells. Complement protein interaction studies with functional readouts, such as reporter assays, to connect observed interactions with transcriptional outcomes. Studies have shown that treatment with the RUNX1 inhibitor Ro 5-3335 partly reverses the synergized effect between RUNX1 and p50, p65, or p105 in NF-κB reporter assays .

How can researchers differentiate between the effects of RUNX1 isoforms in their experimental systems?

Differentiating between RUNX1 isoform effects requires precise experimental designs and specialized reagents. Generate isoform-specific knockdown constructs targeting unique regions of RUNX1B or RUNX1C transcripts, then validate knockdown specificity using isoform-specific qRT-PCR primers and antibodies. Research has demonstrated successful generation of RUNX1C-specific antibodies that recognize the unique 16 amino acid N-terminus of this isoform .

Create isoform-specific overexpression systems using expression vectors containing either RUNX1B or RUNX1C cDNA. Studies have shown that RUNX1B and RUNX1C have opposing effects on P1 and P2 promoter activities when expressed in HeLa cells lacking endogenous RUNX1 . Implement rescue experiments by expressing shRNA-resistant versions of specific isoforms following knockdown of all endogenous RUNX1 to attribute observed phenotypes to particular isoforms.

Design isoform-specific ChIP-seq experiments to identify unique and shared genomic binding sites. Research has demonstrated that both RUNX1B and RUNX1C bind to regions encompassing RUNX1 consensus sites in P1 and P2 promoters, but with different functional outcomes . Employ RNA-seq following isoform-specific manipulation to reveal comprehensive transcriptional networks regulated by each isoform. Studies have identified that in platelets, RUNX1B transcripts correlate positively with F13A1, PCTP, PDE5A, and RAB1B expression, but negatively with MYL9 expression .

How can RUNX1 antibodies be used to investigate its role in cardiovascular disease?

RUNX1 antibodies provide valuable tools for exploring the relationship between RUNX1 isoforms and cardiovascular disease (CVD) pathogenesis. Research has demonstrated that RUNX1C transcripts in whole blood were protective against acute events in CVD patients, while higher expression of RUNX1 target genes F13A1 and RAB31 associated with acute events . When designing studies to investigate these relationships, perform immunohistochemistry with RUNX1 antibodies on cardiovascular tissue samples to assess RUNX1 expression patterns in different vascular cell types during disease progression.

Implement flow cytometry with FITC-conjugated RUNX1 antibodies to quantify RUNX1 expression in circulating blood cells from CVD patients compared to healthy controls. Studies have shown that RUNX1 isoforms differentially regulate downstream genes in platelets, which play critical roles in cardiovascular events . Conduct ChIP-seq experiments in relevant cell types (platelets, endothelial cells, vascular smooth muscle cells) to identify RUNX1 binding sites in genes associated with cardiovascular function and disease.

Develop correlation analyses between RUNX1 isoform expression (detected using isoform-specific antibodies) and clinical parameters or outcomes in cardiovascular disease cohorts. Research has revealed that RUNX1 isoforms B and C regulate downstream genes in a differential manner that associates with acute events in CVD patients . Additionally, perform functional studies in primary cells or relevant cell lines using RUNX1 antibodies to track changes in expression or localization following exposure to cardiovascular disease-relevant stimuli such as inflammatory cytokines, hypoxia, or mechanical stress.

What methodological approaches can be used to study RUNX1's role in inflammation and sepsis?

Investigating RUNX1's role in inflammation and sepsis requires integrated approaches combining antibody-based detection with functional studies. Research has shown that RUNX1 silencing attenuates LPS-induced IL-1β and IL-6 production, while RUNX1 inhibition by Ro 5-3335 protects mice from LPS-induced endotoxic shock . When designing such studies, employ flow cytometry with FITC-conjugated RUNX1 antibodies to track dynamic changes in RUNX1 expression in immune cells following inflammatory stimuli.

Conduct time-course experiments using immunofluorescence microscopy with RUNX1 antibodies to visualize RUNX1 translocation and localization changes during inflammatory activation. Research has demonstrated that RUNX1 mRNA and protein expression levels decrease following LPS stimulation in macrophages . Perform ChIP-seq using RUNX1 antibodies to identify direct transcriptional targets during the inflammatory response, with particular focus on cytokine gene loci.

Implement gain-and-loss-of-function approaches combined with cytokine measurements to establish causality. Studies have shown that RUNX1 overexpression promotes IL-1β and IL-6 production in response to LPS stimulation, while RUNX1 inhibition reduces IL-6 levels in vivo during endotoxic shock . Design co-immunoprecipitation experiments with RUNX1 antibodies followed by mass spectrometry to identify novel interaction partners in inflammatory contexts. Research has identified that RUNX1 interacts with the NF-κB subunit p50 to enhance inflammatory signaling .