RUNX1 Antibody, Biotin conjugated

What is the RUNX1 transcription factor and why is it significant in research?

RUNX1, also known as AML1 (Acute Myeloid Leukemia 1), functions as a key transcription factor forming heterodimeric complexes with CBFB (Core-Binding Factor Beta). This complex recognizes and binds to the core consensus sequence 5'-TGTGGT-3', or rarely 5'-TGCGGT-3', within regulatory regions of target genes . RUNX1 plays an essential role in normal hematopoiesis development and functions through its DNA-binding capabilities via the Runt domain . The significance of RUNX1 in research stems from its critical role in blood cell lineage determination, with expression predominantly in thymus, bone marrow, and peripheral blood tissues, while notably absent in brain and heart tissues . Its involvement in regulatory T-cell function through FOXP3 association and its ability to activate IL2 and IFNG expression while down-regulating TNFRSF18, IL2RA, and CTLA4 in conventional T-cells further underscores its research importance .

What are the primary applications for RUNX1 antibody, biotin conjugated?

The biotin-conjugated RUNX1 antibody serves as a versatile tool across multiple experimental applications. Primary applications include Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB), with recommended dilutions of 1:10,000 for optimal results in both techniques . The antibody can also be utilized in immunohistochemistry (IHC) at a 1:500 dilution . While some RUNX1 antibody variants support additional applications like ChIC/CUT&RUN-seq, immunoprecipitation (IP), flow cytometry, and immunohistochemistry on frozen tissues (IHC-Fr), these applications may be dependent on specific antibody formulations and should be validated before use . The biotin conjugation specifically enhances detection capabilities by allowing for amplification of signal through streptavidin-based detection systems, making it particularly valuable for low-abundance target detection in complex biological samples .

How should researchers properly store and handle RUNX1 antibody, biotin conjugated?

Proper storage and handling of biotin-conjugated RUNX1 antibody is critical for maintaining its functionality and ensuring reproducible experimental results. The antibody should be stored at -20°C for long-term stability . Some formulations may alternatively be stored at -80°C, but researchers should avoid repeated freeze-thaw cycles as this can degrade antibody quality and compromise binding efficiency . The antibody is typically supplied in a stabilization buffer containing 0.03% Proclin 300 as a preservative, 50% glycerol, and 0.01M PBS at pH 7.4 . This formulation helps maintain antibody integrity during storage. When handling the antibody for experimental procedures, it should be thawed completely but gently, kept on ice during use, and returned to storage promptly after aliquoting to minimize exposure to room temperature. Working aliquots may be prepared to avoid repeated freeze-thaw cycles of the stock solution when frequent use is anticipated .

How can researchers validate the specificity of RUNX1 antibody in their experimental system?

Validating antibody specificity is a critical step before conducting experiments with biotin-conjugated RUNX1 antibody. Researchers should implement a multi-step validation process beginning with positive and negative control samples. For positive controls, tissues or cell lines with known RUNX1 expression (such as thymus, bone marrow, or hematopoietic cell lines) should be used, while tissues known to lack RUNX1 expression (brain or heart) serve as negative controls . Western blot analysis should confirm a single band at the expected molecular weight for RUNX1 (approximately 49 kDa, though isoforms may vary). Additional validation can include siRNA knockdown or CRISPR knockout of RUNX1, where signal reduction should be observed with the antibody. For cross-reactivity concerns, particularly when working with antibodies that recognize multiple RUNX family members (RUNX1, RUNX2, RUNX3), researchers should conduct peptide competition assays using the immunogen peptide to confirm binding specificity . When validating for specific applications like ChIP or immunoprecipitation, sequential analysis with multiple antibodies targeting different epitopes of RUNX1 provides robust confirmation of target specificity .

What are the optimal protocols for using biotin-conjugated RUNX1 antibody in Western blotting?

For optimal Western blotting results with biotin-conjugated RUNX1 antibody, researchers should follow a systematic protocol beginning with proper sample preparation. Cell or tissue lysates should be prepared using RIPA buffer supplemented with protease inhibitors, followed by protein quantification to ensure equal loading across samples. Approximately 20-30 μg of total protein should be loaded per lane on a 10-12% SDS-PAGE gel. After electrophoresis, proteins should be transferred to a PVDF membrane (preferred over nitrocellulose for enhanced protein retention). The membrane should be blocked with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature. The RUNX1 biotin-conjugated antibody should be diluted to 1:10,000 in blocking buffer and incubated with the membrane overnight at 4°C with gentle agitation . Following thorough washing with TBST (3-5 times, 5 minutes each), the membrane should be incubated with streptavidin-HRP at 1:5000-1:10000 dilution for 1 hour at room temperature. After additional washing steps, signal development can proceed using enhanced chemiluminescence reagents. When troubleshooting, consider that high background may require more stringent washing or higher antibody dilutions, while weak signals might necessitate longer exposure times or signal amplification techniques .

How can researchers optimize ELISA protocols using biotin-conjugated RUNX1 antibody?

For ELISA optimization with biotin-conjugated RUNX1 antibody, researchers should establish a comprehensive protocol considering multiple parameters. Begin by coating high-binding ELISA plates with capture antibody (typically an unconjugated anti-RUNX1 antibody) at 1-2 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C. After washing with PBS-T (PBS with 0.05% Tween-20), block non-specific binding sites with 2-5% BSA or non-fat dry milk in PBS for 1-2 hours at room temperature. Prepare a standard curve using recombinant RUNX1 protein alongside samples of interest. For detection, the biotin-conjugated RUNX1 antibody should be diluted to 1:10,000 in blocking buffer and incubated for 2 hours at room temperature . Following washing steps, add streptavidin-HRP (1:5000-1:10000) and incubate for 30-60 minutes. After final washing, develop with TMB substrate and stop the reaction with 2N H₂SO₄ before reading absorbance at 450 nm. For sandwich ELISA specifically, ensure the capture and detection antibodies recognize different epitopes to prevent competitive binding. Validation experiments should include determining the linear range of detection and the limit of detection through serial dilutions of positive control samples. Cross-reactivity testing with other RUNX family members may be necessary, especially when using antibodies that might recognize multiple RUNX proteins .

How does RUNX1 function in chromatin remodeling during hematopoietic development?

RUNX1 plays a pivotal role in chromatin remodeling during hematopoietic development, orchestrating blood-cell lineage-specific chromatin priming at surprisingly early developmental stages. Research has demonstrated that RUNX1 initiates the process of chromatin unfolding at critical hematopoietic regulator genes like Pu.1 and Csf1r, marking them for subsequent activation . This mechanism involves RUNX1 binding to its consensus sequences (5'-TGTGGT-3') within regulatory regions of target genes, where it recruits chromatin-modifying enzymes that facilitate the transition from condensed to accessible chromatin states . The molecular process includes histone modifications, particularly H3K4me3 enrichment (associated with active promoters) and H3K27ac accumulation (marking active enhancers). Once RUNX1 has established these initial chromatin alterations, it enables the assembly of stable transcription factor complexes that maintain the active chromatin state in a heritable manner, allowing for continued expression of hematopoietic genes even if RUNX1 expression later diminishes . This establishes a developmental window during which RUNX1 activity is critical; after stable transcription factor circuits form, the direct presence of RUNX1 becomes less essential for maintaining the hematopoietic cell fate, demonstrating the epigenetic memory established through its initial chromatin remodeling activities .

What are the implications of RUNX1 in T-cell development and how can researchers investigate this using biotin-conjugated antibodies?

RUNX1 exhibits multifaceted roles in T-cell development, with significant implications for immune regulation and T-cell lineage commitment. It forms CBF complexes that repress the ZBTB7B transcription factor during cytotoxic (CD8+) T-cell development by binding to RUNX-binding sequences within the ZBTB7B locus, acting as a transcriptional silencer . This binding is essential for recruiting nuclear protein complexes that catalyze epigenetic modifications, establishing epigenetic silencing of ZBTB7B and allowing for cytotoxic T-cell differentiation . Additionally, RUNX1 controls regulatory T-cell (Treg) anergy and suppressive function through association with FOXP3, activating IL2 and IFNG expression while down-regulating TNFRSF18, IL2RA, and CTLA4 in conventional T-cells . RUNX1 also positively regulates RORC expression in T-helper 17 cells .

To investigate these mechanisms using biotin-conjugated RUNX1 antibodies, researchers can employ multiple approaches:

• Chromatin Immunoprecipitation (ChIP): Using biotin-conjugated RUNX1 antibodies with streptavidin-coated magnetic beads to pull down RUNX1-bound chromatin, followed by qPCR or sequencing to identify binding sites within T-cell specific genes.

• Co-immunoprecipitation: Investigating RUNX1 interactions with partners like FOXP3 by precipitating protein complexes with biotin-conjugated RUNX1 antibody followed by western blotting for associated proteins.

• Flow cytometry: Analyzing RUNX1 expression levels across T-cell developmental stages using biotin-conjugated antibodies with fluorochrome-labeled streptavidin.

• Immunofluorescence microscopy: Visualizing nuclear localization and co-localization with other transcription factors during T-cell activation and differentiation .

How can researchers effectively use biotin-conjugated RUNX1 antibody in multiplex detection systems?

Multiplex detection systems offer significant advantages for comprehensive analysis of complex signaling networks involving RUNX1. Biotin-conjugated RUNX1 antibodies can be effectively incorporated into these systems through several optimized approaches. For multiplex immunofluorescence microscopy, researchers should pair the biotin-conjugated RUNX1 antibody with spectrally distinct fluorophore-conjugated streptavidin (e.g., streptavidin-Alexa Fluor 488, 568, or 647) while using primary antibodies from different host species for additional targets to prevent cross-reactivity . Optimal dilutions should be empirically determined, typically starting with 1:500 for tissue sections . For multiplex flow cytometry, intracellular staining protocols must include proper fixation and permeabilization steps, with 0.1% saponin or commercial permeabilization reagents suitable for nuclear transcription factor detection. Sequential staining may be necessary when combining surface and intracellular markers . In multiplex protein detection arrays or bead-based multiplex systems, researchers should validate the absence of cross-reactivity with other analytes in the panel through single-analyte control experiments. Signal amplification can be achieved by incorporating tyramide signal amplification (TSA) after streptavidin-HRP binding. For ChIP-seq or CUT&RUN applications in multiplex genomic studies, biotin-conjugated RUNX1 antibody should be validated for specificity using spike-in controls, and optimized antibody concentrations should be determined to minimize background while maintaining sensitivity . Batch effects can be controlled by including reference samples across experimental runs and incorporating appropriate normalization methods during data analysis .

How can researchers troubleshoot non-specific binding or weak signals when using biotin-conjugated RUNX1 antibody?

When encountering non-specific binding or weak signals with biotin-conjugated RUNX1 antibody, researchers should implement a systematic troubleshooting approach addressing multiple experimental aspects. For non-specific binding issues, first examine blocking conditions by increasing the concentration of blocking agent (5-10% BSA or non-fat dry milk) or trying alternative blockers like normal serum from the secondary reagent species . Incorporating additional washing steps with increased stringency (higher salt concentration or 0.1-0.3% Triton X-100 in wash buffers) can help reduce background signal. Pre-absorbing the antibody with tissue powder from a negative control sample may eliminate cross-reactive antibodies .

For weak signal issues, several strategies can be employed: First, optimize antibody concentration by testing a range of dilutions (e.g., 1:500 to 1:20,000) to identify the optimal signal-to-noise ratio . Antigen retrieval methods should be evaluated for tissue samples, with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) under various heating conditions. Extended primary antibody incubation (overnight at 4°C rather than 1-2 hours at room temperature) often improves signal strength . Signal amplification systems such as tyramide signal amplification (TSA) or poly-HRP streptavidin can enhance detection of low-abundance targets.

To address biotin-specific issues, consider endogenous biotin blocking kits for tissues with high endogenous biotin (liver, kidney, brain) before applying biotin-conjugated antibodies. Finally, sample preparation is crucial - fresh samples generally yield better results than those subjected to prolonged storage, and optimal fixation protocols (4% paraformaldehyde for 10-15 minutes for cells) should be established for consistent results .

How should researchers analyze and interpret RUNX1 binding patterns in chromatin immunoprecipitation experiments?

Analysis and interpretation of RUNX1 binding patterns in chromatin immunoprecipitation (ChIP) experiments require careful consideration of biological context and technical variables. Researchers should begin by establishing robust peak calling parameters, with recommended false discovery rate (FDR) thresholds of ≤0.05 and fold enrichment ≥2-fold over input or IgG control . Motif analysis of RUNX1-bound regions should confirm enrichment of the canonical binding motif (5'-TGTGGT-3' or 5'-TGCGGT-3'), which serves as a quality control measure for antibody specificity .

When analyzing genomic distribution of binding sites, researchers should categorize peaks based on their location relative to transcriptional start sites (promoter regions: ±2kb from TSS; distal regulatory elements: >2kb from TSS), as RUNX1 functions in both proximal promoters and distal enhancers . Integration with expression data is crucial - researchers should correlate ChIP-seq peaks with RNA-seq data from the same cell type to distinguish between functional binding events (associated with gene expression changes) and non-functional interactions. For developmental studies, comparison of RUNX1 binding patterns across different hematopoietic differentiation stages can reveal stage-specific regulatory mechanisms .

Co-localization analysis with other transcription factors and epigenetic marks provides deeper insights into RUNX1 function. Specifically, overlay with H3K4me3 (active promoters), H3K27ac (active enhancers), and binding data for known RUNX1 partners like CBFB can illuminate functional genomic regions . Validation of key binding sites should be performed using ChIP-qPCR with additional biological replicates. For data interpretation, researchers should consider that early chromatin priming by RUNX1 may not immediately correlate with gene expression changes, as demonstrated in studies showing RUNX1-mediated chromatin unfolding precedes transcriptional activation of hematopoietic genes .

How is RUNX1 antibody being utilized in research on hematological malignancies?

RUNX1 antibody applications in hematological malignancy research span diagnostic, prognostic, and mechanistic investigations due to RUNX1's critical role in leukemogenesis. In acute myeloid leukemia (AML), where RUNX1 mutations occur in approximately 10-15% of cases, biotin-conjugated RUNX1 antibodies enable precise immunohistochemical assessment of RUNX1 protein expression patterns in patient samples . These analyses reveal how altered RUNX1 expression correlates with specific cytogenetic abnormalities, particularly the t(8;21) translocation that produces the RUNX1-ETO fusion protein . In mechanistic studies, ChIP-seq applications using RUNX1 antibodies have mapped genome-wide binding alterations of mutant RUNX1 proteins compared to wild-type, identifying dysregulated target genes contributing to leukemogenesis .

For minimal residual disease (MRD) monitoring, multiplexed flow cytometry incorporating RUNX1 antibodies with other hematopoietic markers helps distinguish leukemic cells from normal progenitors . In translational research, RUNX1 antibodies facilitate screening of potential therapeutic compounds targeting the transcriptional complexes formed by RUNX1 and its partners . Co-immunoprecipitation studies using biotinylated RUNX1 antibodies have identified novel protein interactions specific to leukemic contexts, providing new therapeutic targets . Importantly, comparative analysis of RUNX1 binding patterns between healthy and malignant hematopoietic cells has revealed cancer-specific regulatory networks, with RUNX1 showing altered genomic distribution and differential co-factor recruitment in leukemic cells compared to normal counterparts . This research direction offers promising avenues for developing targeted therapies that specifically disrupt aberrant RUNX1-mediated transcriptional programs in hematological malignancies .

What are the latest techniques for studying RUNX1-mediated transcriptional regulation using biotinylated antibodies?

Cutting-edge techniques for studying RUNX1-mediated transcriptional regulation increasingly leverage the high affinity of biotinylated antibodies in combination with advanced genomic and proteomic methodologies. CUT&RUN (Cleavage Under Targets and Release Using Nuclease) represents one of the most significant advances, requiring substantially fewer cells than traditional ChIP-seq while providing higher signal-to-noise ratios . In this technique, biotinylated RUNX1 antibodies bound to protein A-micrococcal nuclease fusion proteins enable targeted DNA cleavage around RUNX1 binding sites, followed by next-generation sequencing of the released fragments. For single-cell applications, technologies like CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) combine biotinylated RUNX1 antibodies with oligonucleotide barcodes, allowing simultaneous analysis of RUNX1 protein levels and transcriptomes in thousands of individual cells .

Proximity ligation assays (PLA) using biotinylated RUNX1 antibodies in combination with antibodies against potential interaction partners enable in situ visualization of protein complexes at specific genomic loci . This approach has revealed cell type-specific RUNX1 co-factor interactions that would be missed in bulk biochemical assays. For studying chromatin looping and three-dimensional genome organization, HiChIP protocols incorporating biotinylated RUNX1 antibodies allow identification of long-range chromatin interactions mediated by RUNX1, providing insights into how distant enhancers regulate RUNX1 target genes .

Nascent RNA capture techniques combined with RUNX1 ChIP-seq have refined our understanding of direct transcriptional targets versus secondary effects, with biotin-streptavidin purification systems enhancing the sensitivity of these approaches . Finally, CRISPR-based epigenome editing systems that recruit RUNX1 to specific genomic loci are being used to dissect the sufficiency of RUNX1 binding for initiating chromatin remodeling and gene activation during hematopoietic specification, complementing the loss-of-function studies that have dominated the field .

How can researchers integrate RUNX1 antibody-based studies with advanced genomic technologies for comprehensive hematopoietic development analysis?

Integrating RUNX1 antibody-based studies with advanced genomic technologies enables comprehensive multi-omics analysis of hematopoietic development. Researchers can implement several strategic approaches to achieve this integration. Sequential ChIP-seq and ATAC-seq on the same cell populations provides correlated maps of RUNX1 binding and chromatin accessibility changes during developmental transitions . This approach reveals how RUNX1 binding precedes and potentially drives accessibility changes at key hematopoietic regulatory elements. Multi-omics single-cell approaches combining protein detection (using biotinylated RUNX1 antibodies with metal-conjugated streptavidin for mass cytometry) and single-cell RNA-seq or ATAC-seq data allow researchers to define precise developmental trajectories where RUNX1 activity is critical .

Spatial transcriptomics technologies can be enhanced with immunofluorescence using biotinylated RUNX1 antibodies to correlate RUNX1 protein localization with gene expression patterns in intact tissues, providing insights into microenvironmental influences on RUNX1 function during development . Time-course experiments using DOX-inducible RUNX1 systems, as described in the literature, combined with temporal ChIP-seq, RNA-seq, and ChIC/CUT&RUN-seq create comprehensive datasets for modeling the dynamic gene regulatory networks controlled by RUNX1 . Computational integration of these multi-omics datasets requires sophisticated bioinformatic approaches, including trajectory inference algorithms, gene regulatory network reconstruction, and machine learning methods to predict RUNX1-dependent developmental decision points .

For mechanistic validation of predictions from integrated analyses, CRISPR perturbation of RUNX1 binding sites identified through ChIP-seq, followed by phenotypic and molecular characterization using antibody-based detection methods, closes the loop between correlation and causation . This multi-layered approach has revealed that RUNX1 orchestrates hematopoietic specification through sequential chromatin priming events that establish self-sustaining transcriptional circuits, a finding with significant implications for understanding both normal development and leukemogenesis .