RUNX1 Antibody, HRP conjugated

What is RUNX1 and why is it significant in research?

RUNX1, also known as AML1-EVI-1 fusion protein or Runt-related transcription factor 1, represents the alpha subunit of Core Binding Factor (CBF), a heterodimeric transcription factor that binds to core elements of many enhancers and promoters. RUNX1 is critically involved in the development of normal hematopoiesis, with chromosomal translocations affecting this gene being well-documented in association with several types of leukemia . RUNX1 forms heterodimeric complexes with CBFB, recognizing the core consensus binding sequence 5'-TGTGGT-3' within regulatory regions via its runt domain . Its significance extends beyond hematopoiesis to sensory neuron diversification and axonal growth, making it a valuable target for immunological detection across multiple research disciplines .

How do I select the appropriate RUNX1 antibody for my western blotting experiments?

When selecting a RUNX1 antibody for western blotting, consider the following criteria:

• Target specificity: Choose antibodies validated for human RUNX1 specificity, such as those that recognize the 55 kDa band characteristic of RUNX1 protein in appropriate cell lysates .

• Format compatibility: For direct detection, select HRP-conjugated primary antibodies; alternatively, use purified primary antibodies with compatible secondary antibodies such as Goat anti-Mouse IgG (H/L):HRP for mouse-derived primaries .

• Validated applications: Confirm the antibody has been specifically validated for western blotting through extensive testing with whole cell lysates .

• Clone consideration: Monoclonal antibodies like clone EF03/2B4 (mouse anti-human) or clone A-2 (mouse IgG1 kappa) offer consistent performance across experiments .

What cell lines serve as positive controls for RUNX1 antibody validation?

The following cell lines have been verified as appropriate positive controls for RUNX1 antibody validation:

These cell lines consistently express RUNX1 and have been utilized in published research to validate antibody specificity and performance . When establishing a new experimental system, it is advisable to include at least one of these lines as a reference control.

How does RUNX1 binding specificity affect experimental design when studying transcriptional regulation?

When designing experiments to study RUNX1-mediated transcriptional regulation:

• Incorporate multiple binding sites: Include all potential RUNX1 binding sites in reporter constructs since isolated sites may not reflect physiological regulation.

• Consider position effects: Examine the spatial relationship between RUNX1 sites and other regulatory elements, particularly GATA sites, as these interactions significantly influence transcriptional outcomes .

• Validate binding capacity: Notably, EMSA analysis has demonstrated that in vitro binding capacity does not necessarily correlate with functional significance in reporter assays, highlighting the importance of multiple methodological approaches .

What considerations should be made when using RUNX1 antibodies in multiplex immunofluorescence applications?

For multiplex immunofluorescence incorporating RUNX1 antibodies:

• Clone selection: Select antibody clones raised in different host species than other primary antibodies in the panel to avoid cross-reactivity.

• Epitope accessibility: Consider that certain fixation methods may mask the RUNX1 epitope, particularly for antibodies targeting the DNA-binding domain.

• Signal amplification: For low-abundance detection, HRP-conjugated antibodies can be used with tyramide signal amplification (TSA) systems, which allow multiple antibodies from the same species to be used sequentially.

• Co-localization analysis: When studying RUNX1 interactions with other transcription factors such as GATA-1 or PU.1, select fluorophores with minimal spectral overlap to enable accurate co-localization analysis .

The rabbit recombinant monoclonal RUNX1/AML1 antibody has been validated for multiple applications, including immunohistochemistry, immunoprecipitation, and flow cytometry, making it suitable for multiplex approaches .

How should I optimize western blot protocols for detecting RUNX1 with HRP-conjugated antibodies?

Optimizing western blot protocols for RUNX1 detection requires attention to several critical parameters:

• Lysate preparation: For optimal RUNX1 detection, prepare whole cell lysates rather than nuclear extracts alone, as validated in comprehensive analyses .

• Protein loading: Load 15-30 μg of total protein per lane, as RUNX1 typically appears as a 55 kDa band on western blots .

• Dilution optimization: For HRP-conjugated primary antibodies, begin with a 1:1000 dilution and adjust based on signal-to-noise ratio .

• Blocking conditions: Use phosphate-buffered saline with 3-5% non-fat milk or BSA for blocking, compatible with the antibody buffer system .

• Detection sensitivity: Employ enhanced chemiluminescence (ECL) substrates with extended signal duration for optimal visualization of RUNX1 bands.

• Stripping and reprobing: Avoid harsh stripping conditions if reusing membranes, as this may damage the RUNX1 epitope.

What are the critical factors in ChIP experiments using RUNX1 antibodies?

When performing Chromatin Immunoprecipitation (ChIP) with RUNX1 antibodies:

• Crosslinking optimization: Use 1% formaldehyde for 10 minutes at room temperature for optimal RUNX1-DNA crosslinking, as longer durations may adversely affect epitope recognition.

• Sonication parameters: Adjust sonication conditions to generate chromatin fragments of 200-500 bp, ideal for RUNX1 binding site resolution.

• Antibody selection: Choose ChIP-validated antibodies specifically tested for this application, such as the rabbit recombinant monoclonal RUNX1 antibody .

• Positive control regions: Include the following validated RUNX1 binding regions as positive controls:

• Quantification method: Use qPCR rather than endpoint PCR for more accurate quantification of enrichment at RUNX1 binding sites.

Why might I observe multiple bands when using RUNX1 antibodies in western blotting?

Multiple bands in RUNX1 western blots can result from several biological and technical factors:

• Isoform detection: RUNX1 has three transcript variants encoding different isoforms, which may appear as distinct bands . Specifically:

  • AML1c (full-length): ~49-55 kDa

  • AML1b: ~40-45 kDa

  • AML1a: ~25-30 kDa

• Post-translational modifications: Phosphorylation, ubiquitination, and SUMOylation of RUNX1 can alter migration patterns.

• Proteolytic processing: Sample preparation conditions may lead to partial degradation, generating truncated forms.

• Antibody cross-reactivity: Some antibodies may cross-react with other RUNX family members (RUNX2, RUNX3) due to sequence homology, particularly in the runt domain .

To distinguish between these possibilities:

• Use positive control lysates with known RUNX1 expression patterns

• Compare results across multiple antibody clones targeting different epitopes

• Include protease inhibitors in lysis buffers

• Test RUNX1-knockout or knockdown samples as negative controls

How can I distinguish between binding specificity and functional relevance in RUNX1 transcriptional studies?

Distinguishing between binding capacity and functional relevance in RUNX1 studies requires a multi-faceted approach:

• Combined EMSA and reporter assays: Research has revealed discrepancies between in vitro binding capacity (EMSA) and functional significance (reporter assays). For example, the distal RUNX1 site in the CCR3 gene showed stronger binding in EMSA but had less impact on reporter activity than the proximal site .

• Mutagenesis analysis: Systematically mutate individual and combinations of RUNX1 binding sites in reporter constructs to assess their functional contributions. Include:

  • Single-site mutations

  • Multiple-site mutations

  • Mutations in combination with other transcription factor binding sites (e.g., GATA-1, PU.1)

• siRNA knockdown: Transfect cells with RUNX1 siRNA alongside reporter constructs to quantify the direct contribution of RUNX1 to transcriptional activity. Note that RUNX1 knockdown in CCR3 studies produced a modest 22% reduction in reporter activity compared to 60% for GATA-1 knockdown .

• Chromatin conformation assays: Employ chromosome conformation capture (3C) techniques to verify physical interactions between RUNX1-bound enhancers and promoters in their native genomic context.

How do RUNX1 antibodies contribute to understanding hematopoietic differentiation and leukemogenesis?

RUNX1 antibodies have become instrumental in elucidating the mechanisms of hematopoietic differentiation and leukemogenesis:

• Lineage commitment studies: RUNX1 is essential for generating hematopoietic lineages, with antibodies helping to track its expression during differentiation . Studies have shown that RUNX1 deficiency dramatically decreases basophil populations while having less impact on neutrophils and eosinophils, highlighting its lineage-specific roles .

• Chromosomal translocation detection: RUNX1 antibodies can identify fusion proteins resulting from chromosomal translocations (e.g., RUNX1-ETO in t(8;21) acute myeloid leukemia) .

• Regulatory T-cell function: RUNX1 antibodies have revealed its role in controlling anergy and suppressive functions of regulatory T-cells through association with FOXP3, activating IL2 and IFNG expression while down-regulating TNFRSF18, IL2RA, and CTLA4 in conventional T-cells .

• Cooperative transcription factor networks: Combined with antibodies against other factors like PU.1 and GATA-1, RUNX1 antibodies have demonstrated how these factors work together to regulate lineage-specific genes in eosinophil development .

What are the current technical limitations in using HRP-conjugated RUNX1 antibodies for chromatin studies?

Several technical limitations affect the application of HRP-conjugated RUNX1 antibodies in chromatin studies:

• Epitope accessibility: The HRP conjugation can reduce antibody access to RUNX1 epitopes in condensed chromatin structures.

• Cross-linking interference: The bulky HRP moiety may interfere with antibody-antigen interactions in formaldehyde-fixed samples.

• Resolution limitations: Direct HRP conjugation prevents the signal amplification achieved through secondary antibody binding, potentially reducing sensitivity for low-abundance binding sites.

• Compatibility with sequential ChIP: HRP-conjugated antibodies are generally unsuitable for sequential ChIP (re-ChIP) protocols designed to identify co-occupancy of multiple factors.

• Methodological alternatives: For chromatin studies, researchers should consider:

  • Using unconjugated primary antibodies with HRP-conjugated secondary antibodies

  • CUT&RUN protocols with protein A/G-conjugated peroxidases instead of direct HRP conjugation

  • ChIP-sequencing with non-conjugated antibodies for genome-wide binding analysis