brd9 Antibody

What is BRD9 and why is it significant in epigenetic research?

BRD9 is a bromodomain-containing protein that functions as a chromatin reader by recognizing and binding to acetylated lysine residues on histones. It plays a critical role in chromatin remodeling and transcriptional regulation . BRD9 is also known by alternative names including LAVS3040, PRO9856, and rhabdomyosarcoma antigen MU-RMS-40.8 . Structurally, the protein has a molecular weight of approximately 67 kDa .

The significance of BRD9 has been highlighted in recent studies demonstrating its critical role in cancer biology, particularly in acute myeloid leukemia (AML) where it has been identified as a potential therapeutic target . BRD9 has been shown to bind enhancer regions in a cell type-specific manner, regulating cell type-related processes and maintaining leukemic states through a previously undescribed BRD9-STAT5 signaling axis .

What applications are BRD9 antibodies typically used for in research?

BRD9 antibodies are versatile research tools applicable across multiple experimental techniques. According to available data, these antibodies have demonstrated utility in:

For optimal results in immunohistochemistry applications, antigen retrieval with TE buffer (pH 9.0) is recommended, though citrate buffer (pH 6.0) can serve as an alternative .

How do I select the appropriate BRD9 antibody for my specific research needs?

When selecting a BRD9 antibody, consider these critical parameters:

• Specific epitope recognition: Determine whether you need an antibody targeting the N-terminal, C-terminal, or bromodomain regions. Some antibodies are designed to recognize specific regions, such as the N-terminal domain .

• Cross-reactivity profile: Evaluate the antibody's reactivity with orthologs from relevant species. While most BRD9 antibodies recognize human samples, some show cross-reactivity with mouse, rat, and other species .

• Validated applications: Ensure the antibody has been validated for your intended application. For instance, if conducting ChIP-seq experiments, select an antibody specifically validated for chromatin immunoprecipitation .

• Clone type: Consider whether a monoclonal or polyclonal antibody better suits your experimental needs. Monoclonal antibodies offer high specificity for particular epitopes, while polyclonal antibodies may provide stronger signals by recognizing multiple epitopes .

• Literature validation: Prioritize antibodies with published validation in peer-reviewed research, particularly for techniques similar to your planned experiments .

What is the optimal protocol for using BRD9 antibodies in Western blotting?

For optimal Western blot results with BRD9 antibodies, follow this evidence-based protocol:

• Sample preparation:

  • Prepare cell or tissue lysates using appropriate lysis buffers containing protease inhibitors

  • For nuclear proteins like BRD9, nuclear extraction protocols yield better results than whole-cell lysates

• SDS-PAGE separation:

  • Load 20-50 μg of protein per lane

  • Use 8-10% gels for optimal resolution of the 67 kDa BRD9 protein

• Transfer and blocking:

  • Transfer to PVDF or nitrocellulose membranes

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Antibody incubation:

  • Dilute primary BRD9 antibody at 1:500-1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Wash membranes thoroughly with TBST (3-5 washes, 5 minutes each)

  • Incubate with appropriate HRP-conjugated secondary antibody

• Detection and expected results:

  • Detect using enhanced chemiluminescence

  • Expected molecular weight: 67 kDa (major band)

  • Additional bands may correspond to known isoforms (23, 26, 53, 56, and 61 kDa)

For cell line validation, HeLa and HEK-293 cells have been confirmed to express detectable levels of BRD9 protein .

How should I optimize immunoprecipitation experiments using BRD9 antibodies?

For successful BRD9 immunoprecipitation experiments, implement this methodological approach:

• Nuclear extraction preparation:

  • Lyse nuclear pellets in buffer C (20 mM HEPES pH 7.6, 20% glycerol, 0.42 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA) containing protease inhibitors

  • Dialyze against buffer C-100 (20 mM HEPES pH 7.6, 20% glycerol, 0.2 mM EDTA, 100 mM KCl, 1.5 mM MgCl₂)

• Antibody coupling:

  • Couple 2-5 μg of BRD9 antibody to protein A/G magnetic beads

  • For epitope-tagged BRD9 experiments, use tag-specific antibodies (e.g., HA or V5)

• Immunoprecipitation:

  • Incubate antibody-coupled beads with dialyzed nuclear extracts containing 250 U of benzonase

  • Maintain interaction for 3 hours at 4°C with rotation

  • Perform thorough washes (at least 3-5) with buffer C-100

• Elution strategies:

  • For denaturing conditions: use 1× LDS buffer

  • For native conditions: use 1 mg/mL peptide elution (HA or V5 peptide for tagged constructs)

• Controls and validation:

  • Include IgG control to identify non-specific binding

  • Validate IP efficiency by comparing input, unbound, and eluted fractions by Western blot

This protocol has been successfully utilized to investigate BRD9 interactions in chromatin regulation studies .

What are the best practices for immunohistochemical detection of BRD9 in tissue samples?

For optimal immunohistochemical detection of BRD9 in tissue samples:

• Tissue preparation and fixation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin

  • Section at 4-5 μm thickness onto positively charged slides

• Antigen retrieval:

  • Primary recommendation: Use TE buffer (pH 9.0) for heat-induced epitope retrieval

  • Alternative method: Citrate buffer (pH 6.0)

  • Heat in pressure cooker or microwave until boiling, then maintain for 10-20 minutes

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% H₂O₂

  • Block non-specific binding with serum-free protein block

  • Incubate with BRD9 antibody at 1:50-1:500 dilution

  • Optimal incubation: Overnight at 4°C or 1-2 hours at room temperature

• Detection and visualization:

  • Use polymer-based detection systems for superior sensitivity

  • Develop with DAB and counterstain with hematoxylin

  • Apply aqueous mounting medium for long-term preservation

• Controls and interpretation:

  • Include positive control (human colon cancer tissue shows reliable BRD9 expression)

  • Include negative controls (primary antibody omission)

  • Expect primarily nuclear staining pattern with potential cytoplasmic signal in some cell types

Optimize antibody concentration based on your specific tissue type and fixation conditions.

How does BRD9 function in the context of chromatin regulation and what methodologies best capture this activity?

BRD9 functions as a component of the SWI/SNF chromatin remodeling complex, specifically as part of the non-canonical BAF complex. To investigate its chromatin regulatory functions:

• Genome-wide binding profile analysis:

  • Implement ChIP-seq using validated BRD9 antibodies

  • Analysis reveals that BRD9 binds enhancer regions in a cell type-specific manner, regulating cell type-related processes

  • Integrate with histone modification ChIP-seq data (H3K27ac, H3K4me1) to confirm enhancer associations

• Transcriptional impact assessment:

  • Compare RNA-seq data between BRD9 inhibitor-treated (I-BRD9) and control cells

  • Analyze BRD9 knockdown/knockout models using CRISPR-Cas9 or RNAi approaches

  • Key finding: BRD9 regulates SOCS3 expression, which impacts STAT5 pathway activation in leukemic cells

• Protein-protein interaction studies:

  • Employ proximity ligation assays to detect endogenous BRD9 interactions

  • Use Co-IP followed by mass spectrometry to identify novel binding partners

  • BioID or APEX2 proximity labeling can map the BRD9 interaction network in living cells

• Chromatin accessibility analysis:

  • Integrate ATAC-seq or DNase-seq with BRD9 binding data

  • This approach reveals how BRD9 influences chromatin structure at target loci

The BRD9-STAT5 regulatory axis identified through these methods represents a novel mechanism in leukemia maintenance, suggesting therapeutic potential through BRD9 targeting .

What are the considerations when using chemical probes like I-BRD9 alongside BRD9 antibodies in research?

When combining chemical probes with antibody-based approaches:

• Selectivity profiles and experimental design:

  • I-BRD9 offers >700-fold selectivity over BET family bromodomains and >70-fold selectivity against a panel of 34 other bromodomains

  • For reliable results, maintain probe concentrations below the selectivity threshold (typically ≤1 μM for I-BRD9)

  • Include BRD7 considerations, as I-BRD9 shows only 200-fold selectivity over this closely related bromodomain

• Target engagement validation:

  • Implement cellular thermal shift assays (CETSA) with BRD9 antibodies to confirm target engagement

  • Nanobret assays can quantitatively measure compound-target interactions in cells (I-BRD9 pIC₅₀ = 6.8 in NanoBRET)

• Downstream signaling analysis:

  • Use phospho-specific antibodies for STAT5 to monitor the BRD9-STAT5 axis

  • Quantify SOCS3 expression changes (qPCR, Western blot) as a functional readout

  • Compare antibody-based knockdown results with small molecule inhibition profiles

• Physicochemical properties and experimental variables:

  • Consider I-BRD9's properties: ChromLogD (pH 7.4) of 3.7, high aqueous solubility (359 μM), and high membrane permeability (210 nm/s)

  • These properties influence experimental design, including dosing, timing, and washout periods

• Control compounds:

  • Include negative control compounds with similar structure but lacking BRD9 activity

  • For mechanistic studies, compare I-BRD9 effects with genetic perturbation of BRD9

This integrated approach has identified BRD9 inhibitor-sensitive genes involved in immune function and cancer, providing insights into BRD9 bromodomain biological roles .

How can I distinguish between BRD9 and the highly homologous BRD7 in my experiments?

Distinguishing between BRD9 and its close homolog BRD7 requires careful experimental design:

• Antibody selection strategies:

  • Choose antibodies raised against regions with minimal sequence homology between BRD9 and BRD7

  • Validate antibody specificity using BRD9 and BRD7 knockout cell lines or siRNA-treated samples

  • Consider using epitope-tagged constructs when studying exogenous expression

• Specific chemical probe utilization:

  • I-BRD9 demonstrates approximately 200-fold selectivity for BRD9 over BRD7

  • At concentrations below 500 nM, I-BRD9 primarily inhibits BRD9 with minimal BRD7 effects

  • Compare with dual BRD7/9 inhibitors to distinguish shared versus unique functions

• Genomic approaches:

  • Design PCR primers, CRISPR guides, or RNAi sequences targeting unique regions

  • For ChIP-seq or CUT&RUN experiments, validate antibody specificity with knockout controls

  • Implement spike-in controls for quantitative comparison between experiments

• Proteomic validation:

  • Use isoform-specific peptides for targeted mass spectrometry

  • Analyze protein complexes to identify unique interaction partners for BRD9 versus BRD7

  • Consider protein-fragment complementation assays with specific domains

• Functional readouts:

  • BRD9, but not BRD7, regulates the SOCS3-STAT5 axis in leukemic cells

  • Design functional assays based on these unique downstream targets

These approaches allow researchers to delineate specific functions of these highly homologous bromodomain proteins while minimizing cross-reactivity issues.

What are common issues with BRD9 antibody experiments and how can they be resolved?

Researchers frequently encounter these challenges when working with BRD9 antibodies:

• Nonspecific bands in Western blot:

  • Problem: Additional unexpected bands appear alongside the 67 kDa BRD9 band

  • Resolution:

    • Validate using BRD9 knockout controls

    • Consider the presence of multiple isoforms (23, 26, 53, 56, and 61 kDa)

    • Optimize antibody dilution (recommended range: 1:500-1:2000)

    • Increase blocking stringency with 5% BSA instead of milk

• Weak or absent signal in IHC/IF:

  • Problem: Poor or inconsistent staining in tissue samples

  • Resolution:

    • Compare TE buffer (pH 9.0) versus citrate buffer (pH 6.0) for antigen retrieval

    • Extend primary antibody incubation time (overnight at 4°C)

    • Implement signal amplification systems (tyramide signal amplification)

    • Test multiple antibody clones targeting different epitopes

• Immunoprecipitation inefficiency:

  • Problem: Poor recovery of BRD9 protein in IP experiments

  • Resolution:

    • Use nuclear extraction protocols optimized for chromatin-associated proteins

    • Include benzonase (250 U) to release chromatin-bound proteins

    • Cross-link proteins before lysis for transient interactions

    • Optimize antibody-to-bead and antibody-to-lysate ratios

• Cell type variation:

  • Problem: Inconsistent results across different cell types

  • Resolution:

    • Verify BRD9 expression levels (HeLa and HEK-293 are confirmed positive controls)

    • Adjust lysis conditions based on cell type (harsher conditions may be needed for certain lines)

    • Consider isoform expression differences between tissues

• Reproducibility issues:

  • Problem: Variable results between experiments

  • Resolution:

    • Standardize cell culture conditions (confluence, passage number)

    • Prepare fresh antibody dilutions for each experiment

    • Document lot-to-lot variation and validate each new antibody lot

Implementing these solutions can significantly improve experimental outcomes with BRD9 antibodies.

How do I interpret conflicting results between antibody-based detection and genomic data for BRD9?

When faced with discrepancies between antibody-based and genomic approaches:

• Expression level versus activity assessment:

  • Protein abundance (detected by antibodies) may not correlate with genomic binding or activity

  • Compare ChIP-seq signal intensity with protein levels detected by Western blot

  • Implement activity-based assays (e.g., bromodomain-histone peptide binding assays)

• Post-translational modification considerations:

  • Some antibodies may preferentially detect specific post-translationally modified forms of BRD9

  • Phosphorylation, acetylation, or ubiquitination can affect epitope accessibility

  • Use modification-specific antibodies or mass spectrometry to identify relevant modifications

• Isoform-specific effects:

  • BRD9 has multiple isoforms (67, 23, 26, 53, 56, and 61 kDa)

  • Genomic methods may not distinguish isoform-specific functions

  • Design isoform-specific primers for qPCR validation of RNA-seq data

  • Use isoform-specific antibodies when available

• Experimental context differences:

  • Cell fixation (for ChIP/CUT&RUN) versus native conditions (for Western blot/IP)

  • Chromatin state and accessibility variations between experimental approaches

  • Time-dependent changes in BRD9 localization or expression

• Integrated validation approach:

  • Implement orthogonal methods (e.g., CUT&RUN, CUT&Tag, ATAC-seq)

  • Use genetic approaches (CRISPR knockout/knockdown) alongside antibody detection

  • Chemical genetic strategies (I-BRD9 treatment, degron approaches) provide functional validation

This systematic evaluation helps resolve apparent conflicts between different experimental approaches.

What controls are essential for validating BRD9 antibody specificity in various applications?

To ensure experimental rigor with BRD9 antibodies, implement these essential controls:

These validation strategies collectively establish antibody specificity and reliability.

How might BRD9 antibodies contribute to understanding cancer pathogenesis and therapeutic development?

BRD9 antibodies offer several promising avenues for cancer research and therapeutic development:

• Biomarker development and patient stratification:

  • BRD9 expression and localization patterns may predict therapy response

  • Develop immunohistochemical scoring systems using validated antibodies

  • Correlation between BRD9 status and clinical outcomes in different cancer types

  • Focus on AML where BRD9 has been identified as critical for leukemic maintenance

• Mechanism-based combination therapy approaches:

  • BRD9-STAT5 axis targeting through dual inhibition strategies

  • Use antibodies to monitor pathway modulation during treatment

  • Identify synergistic combinations with BRD9 inhibitors through antibody-based pathway analysis

  • Evaluate effects on resistance mechanisms in longitudinal samples

• Protein degradation therapeutic monitoring:

  • Targeted BRD9 degradation has shown promising results in reversing oncogenic gene expression

  • Antibodies can monitor degradation efficiency in patient samples

  • Development of companion diagnostics for BRD9-targeting therapies

  • Quantitative assessment of residual protein in resistance settings

• Cell type-specific functions:

  • BRD9 binds enhancer regions in a cell type-specific manner

  • Multiplex immunofluorescence with lineage markers can map BRD9 expression across tumor ecosystems

  • Spatial transcriptomics integration with antibody-based detection

  • Single-cell approaches to understand heterogeneity in BRD9 dependency

• Post-translational modification landscape:

  • Develop modification-specific antibodies (phospho, acetylation, etc.)

  • Map how cancer-relevant signaling pathways regulate BRD9 function

  • Correlate modifications with chromatin binding patterns and transcriptional outcomes

These research directions position BRD9 antibodies as critical tools for both fundamental cancer biology and translational applications in precision oncology.

What emerging technologies might enhance BRD9 antibody-based research in the near future?

Several emerging technologies promise to revolutionize BRD9 antibody applications:

• Spatial multi-omics integration:

  • Combining antibody-based spatial proteomics with spatial transcriptomics

  • Technologies like 10X Visium integrated with immunofluorescence

  • Resolving BRD9 protein localization with target gene expression in tissue context

  • Correlation with chromatin accessibility maps in the same samples

• Proximity-based interactome mapping:

  • TurboID or APEX2 fusion proteins for in vivo biotinylation of BRD9 interactors

  • Antibody-guided proximity labeling in fixed tissues

  • Single-cell interactome analysis using antibody-based sorting followed by mass spectrometry

  • Mapping dynamic changes in the BRD9 interactome during cellular differentiation or treatment

• Live-cell imaging innovations:

  • Antibody fragments (nanobodies) for live-cell tracking of endogenous BRD9

  • Fluorescent timer fusions to monitor BRD9 protein turnover

  • FRET-based sensors for BRD9 conformational changes upon inhibitor binding

  • Optogenetic control of BRD9 recruitment to specific genomic loci

• Single-molecule approaches:

  • Super-resolution microscopy with BRD9 antibodies

  • Single-molecule tracking to measure BRD9 chromatin binding kinetics

  • Visualizing individual BRD9-containing complexes on chromatin fibers

  • Correlative light-electron microscopy for ultrastructural localization

• Liquid biopsy applications:

  • Detection of BRD9 protein in circulating tumor cells or extracellular vesicles

  • Development of highly sensitive assays for minimal residual disease monitoring

  • Evaluation of BRD9 as a circulating biomarker for treatment response

  • Integration with cell-free DNA analysis for comprehensive tumor monitoring

These technological advances will expand the utility of BRD9 antibodies beyond conventional applications, enabling more sophisticated investigations of BRD9 biology in health and disease.

How can researchers effectively combine BRD9 antibody approaches with functional genomics to advance epigenetic research?

Integrating BRD9 antibody approaches with functional genomics creates powerful research synergies:

• Comprehensive epigenetic profiling strategies:

  • Combine BRD9 ChIP-seq with histone modification mapping (H3K27ac, H3K4me1/3)

  • Integrate with chromatin accessibility data (ATAC-seq, DNase-seq)

  • Correlate with three-dimensional chromatin organization (Hi-C, Micro-C)

  • Create multi-modal epigenetic atlases across cell types and conditions

• CRISPR screening with antibody-based readouts:

  • CRISPR activation/interference screens targeting BRD9-bound enhancers

  • High-content imaging with BRD9 antibodies as phenotypic readouts

  • Pooled CRISPR screens followed by antibody-based cell sorting

  • Domain-focused mutagenesis to map functional regions of BRD9

• Single-cell multi-modal analysis:

  • CITE-seq/REAP-seq with BRD9 antibodies for protein detection

  • Integration with single-cell ATAC-seq and RNA-seq

  • Trajectory analysis to understand BRD9 dynamics during cellular transitions

  • Deconvolution of heterogeneous responses to BRD9 inhibition

• Machine learning integration:

  • Predictive modeling of BRD9 binding sites based on underlying sequence features

  • Classification of BRD9-dependent versus independent regulatory elements

  • Integration of antibody-based high-content imaging data with genomic datasets

  • Network analysis to position BRD9 within broader epigenetic regulatory circuits

• Therapeutically relevant functional assays:

  • CRISPR-based genetic interaction maps with BRD9 perturbation

  • Chemical-genetic screening to identify synthetic lethal interactions

  • Antibody-based monitoring of compensatory mechanisms after BRD9 inhibition

  • Ex vivo patient sample testing with functional genomic readouts