BRD3 Antibody

What is BRD3 and why is it important in epigenetic research?

BRD3 (Bromodomain Containing 3) is a critical chromatin reader that recognizes and binds acetylated histones, thereby controlling gene expression and remodeling chromatin structures . As a member of the BET (Bromodomain Extra Terminal) family, BRD3 contains two tandem bromodomains (BD1 and BD2) and an extraterminal (ET) domain . BRD3 recruits transcription factors and coactivators to target gene sites and activates RNA polymerase II machinery for transcriptional elongation . Its importance in epigenetic research stems from its ability to bind acetylated lysine residues on histones H2A, H2B, H3, and H4, as well as non-histone acetylated proteins like GATA1 and GATA2 . Recent studies have also implicated BRD3 in inflammatory responses, stress adaptation, and various disease mechanisms, making it a valuable target for research into transcriptional regulation and potential therapeutic interventions .

What applications are BRD3 antibodies commonly used for in research?

BRD3 antibodies are employed in multiple research techniques including:

• Western Blotting (WB): Used for detecting and quantifying BRD3 protein expression with recommended dilutions ranging from 1:500 to 1:2000

• Chromatin Immunoprecipitation (ChIP and ChIP-Seq): Critical for studying BRD3 genomic occupancy and its interactions with chromatin

• Immunoprecipitation (IP): For isolating BRD3 protein complexes and studying protein-protein interactions

• Immunohistochemistry (IHC): For visualizing BRD3 expression in tissue samples

• Immunofluorescence/Immunocytochemistry (IF/ICC): For subcellular localization studies

• Flow Cytometry: For analyzing BRD3 expression in cell populations

Most commercially available BRD3 antibodies show reactivity with human samples, with some also validated for mouse and rat models .

How do I select the appropriate BRD3 antibody for my specific research application?

Selection should be based on:

• Application compatibility: Verify the antibody has been validated for your specific application (WB, ChIP, IF, etc.)

• Species reactivity: Ensure compatibility with your experimental model organism (human, mouse, rat)

• Antibody type: Consider whether polyclonal or monoclonal is more appropriate:

  • Polyclonal: Offers higher sensitivity but potentially lower specificity; useful for detecting low abundance targets

  • Monoclonal: Provides higher specificity and consistency between lots; superior for quantitative applications

• Epitope location: Some antibodies target N-terminal regions while others target C-terminal regions; this matters when studying specific domains or truncated variants

• Validation data: Review published literature citing the antibody and examine manufacturer validation data including positive controls

• Protocol compatibility: Consider buffer compatibility and validated dilution ranges for your specific protocol

For ChIP applications specifically, seek antibodies with demonstrated ChIP-grade quality and published ChIP-seq datasets .

What are the optimal conditions for using BRD3 antibodies in ChIP experiments?

For successful BRD3 ChIP experiments:

• Antibody amount: Typically 1-10 μL per ChIP reaction, with Cell Signaling Technology recommending 1:50 dilution for their BRD3 (E3D5N) Rabbit mAb

• Cross-linking conditions: Standard 1% formaldehyde for 10 minutes at room temperature is typically sufficient for BRD3 ChIP

• Sonication parameters: Aim for chromatin fragments of 200-500 bp for optimal BRD3 binding site resolution

• Controls: Include:

  • Input control (non-immunoprecipitated chromatin)

  • IgG negative control (same species/isotype as BRD3 antibody)

  • Positive control loci (known BRD3 target genes)

• Washing stringency: Multiple washes with increasing salt concentration to reduce background

• Elution and reversal: Standard elution buffers and overnight reversal of cross-linking at 65°C

When designing primers for ChIP-qPCR validation, target regions with known histone acetylation marks, particularly H3K18ac, which has been shown to interact with BRD3 .

How should I design experiments to study BRD3's interaction with transcription factors like GATA1?

Based on published methodologies , a comprehensive approach includes:

• Co-immunoprecipitation (Co-IP):

  • Use anti-BRD3 antibody to pull down protein complexes

  • Western blot for associated transcription factors (e.g., GATA1)

  • Include acetylation inhibitors as negative controls to verify acetylation-dependent interactions

• Sequential ChIP (Re-ChIP):

  • First ChIP with anti-BRD3 antibody

  • Second ChIP with anti-GATA1 antibody

  • Analyze enrichment at common target genes

• Genome-wide occupancy analysis:

  • Perform parallel ChIP-seq for BRD3 and transcription factors

  • Compare binding profiles using bioinformatic approaches

  • Focus on:

    • Co-occupied sites

    • Sites with differential occupancy under various conditions

    • Correlation with histone acetylation marks

• Functional validation:

  • BRD3 knockdown/knockout followed by transcription factor ChIP

  • Analysis of target gene expression

  • Use of BET inhibitors to disrupt interactions

This experimental design has successfully revealed that BRD3 and GATA1 physically interact in an acetylation-dependent manner, and that BRD3 occupies most GATA1-occupied regulatory DNA elements .

What controls should be included when validating a new BRD3 antibody for specificity?

A thorough validation approach should include:

• Molecular controls:

  • BRD3 knockout/knockdown cells or tissues as negative controls

  • BRD3 overexpression systems as positive controls

  • Testing against recombinant BRD3 protein

• Cross-reactivity assessment:

  • Test against other BET family members (BRD2, BRD4, BRDT)

  • Analysis of pattern consistency across multiple cell lines

• Multiple technique validation:

  • Western blot: Verify single band of expected molecular weight (typically observed at 95-105 kDa)

  • IP followed by mass spectrometry identification

  • Immunofluorescence pattern consistent with known subcellular localization

• Epitope blocking experiments:

  • Pre-incubate antibody with immunizing peptide

  • Should eliminate specific signal in all applications

• Alternative antibody comparison:

  • Test multiple antibodies targeting different epitopes

  • Results should show consistent patterns

When publishing, include complete validation data and antibody catalog information to ensure reproducibility.

Why might I observe different molecular weights for BRD3 in Western blots?

The calculated molecular weight of BRD3 is approximately 61 kDa (556 amino acids), yet observed molecular weights typically range from 95-105 kDa . This discrepancy may result from:

• Post-translational modifications:

  • Phosphorylation: BRD3 contains multiple potential phosphorylation sites

  • Acetylation: As an acetyl-lysine binding protein, BRD3 itself may be acetylated

  • SUMOylation or ubiquitination: Can significantly increase apparent molecular weight

• Protein isoforms:

  • Alternative splicing generating different BRD3 variants

  • Tissue or cell type-specific isoform expression

• Technical factors:

  • Incomplete denaturation: Particularly common with chromatin-associated proteins

  • Running buffer composition affecting mobility

  • Gel percentage affecting resolution of higher molecular weight proteins

• Experimental conditions:

  • Sample preparation method (lysis buffers, detergent concentration)

  • Reducing vs. non-reducing conditions

  • Heat denaturation time and temperature

To address these variations, include positive controls with known BRD3 expression, optimize sample preparation protocols, and compare results with multiple BRD3 antibodies targeting different epitopes .

How can I improve signal-to-noise ratio in BRD3 ChIP experiments?

Based on reported challenges with BRD3 ChIP signal-to-noise ratios , consider these optimization strategies:

• Antibody considerations:

  • Test multiple BRD3 antibodies; monoclonal antibodies often provide better specificity

  • Titrate antibody concentration (too much can increase background)

  • Pre-clear chromatin with protein A/G beads before antibody addition

• Cross-linking optimization:

  • Adjust formaldehyde concentration (typically 0.75-1.5%)

  • Test dual cross-linking with additional agents (DSG, EGS) for improved protein-protein cross-linking

  • Fine-tune cross-linking time (8-12 minutes typically optimal)

• Sonication parameters:

  • Ensure consistent fragment size (200-500bp)

  • Avoid over-sonication which can damage epitopes

  • Verify sonication efficiency by gel electrophoresis before proceeding

• Washing conditions:

  • Increase wash stringency with higher salt concentrations

  • Add detergents (0.1% SDS, 1% Triton X-100) to reduce non-specific binding

  • Increase number of washes while maintaining gentle agitation

• Data analysis approaches:

  • Use appropriate peak-calling algorithms optimized for transcription factors

  • Include input normalization and IgG controls

  • Consider using spike-in controls for quantitative normalization

These optimizations address the challenges noted in the literature where "the signal-to-noise ratio of the Brd3A ChIP was not as high as that of GATA1" .

What are common pitfalls when using BRD3 antibodies in immunohistochemistry, and how can they be avoided?

Common IHC challenges with BRD3 antibodies include:

• Antigen retrieval issues:

  • Problem: Insufficient epitope exposure

  • Solution: Compare citrate buffer (pH 6.0) vs. TE buffer (pH 9.0); most BRD3 antibodies perform better with higher pH buffers

• Non-specific staining:

  • Problem: Background signal especially in highly vascularized tissues

  • Solution: Implement additional blocking steps (avidin/biotin blocking, protein block); optimize antibody dilution (typically 1:50-1:500)

• Fixation artifacts:

  • Problem: Overfixation masking epitopes or underfixation causing tissue degradation

  • Solution: Standardize fixation time (18-24 hours in 10% neutral buffered formalin is typically optimal)

• Variable expression levels:

  • Problem: Low endogenous BRD3 expression in some tissues

  • Solution: Use amplification systems (e.g., tyramide signal amplification); include positive control tissues with known BRD3 expression

• Cross-reactivity with other BET proteins:

  • Problem: Similar sequence homology between BRD2, BRD3, and BRD4

  • Solution: Validate antibody specificity with peptide competition assays and BRD3-knockout controls

For optimal results, follow tissue-specific recommendations for antigen retrieval and include comprehensive controls in each staining batch .

How should I interpret BRD3 ChIP-seq data in relation to gene activation versus repression?

Based on genome-wide occupancy studies , BRD3 ChIP-seq data interpretation requires nuanced analysis:

The genome-wide analysis by Lamonica et al. revealed that "Brd3 is versatile in nature, occupying both transcriptionally active and inactive euchromatin" , highlighting the importance of comprehensive data integration for accurate interpretation.

How can I differentiate between direct and indirect effects of BRD3 inhibition in functional studies?

To distinguish direct from indirect effects:

• Temporal analysis:

  • Monitor gene expression changes at multiple early time points after BRD3 inhibition

  • Direct targets typically show rapid expression changes (within hours)

  • Indirect effects accumulate at later time points

• Dose-response relationships:

  • Direct targets often show dose-dependent responses to BRD3 inhibitors

  • Compare with BRD3 knockdown/knockout phenotypes

• Genomic occupancy correlation:

  • Integrate ChIP-seq data to identify genes with BRD3 binding

  • Direct targets should have BRD3 occupancy at regulatory regions

  • Use of complementary techniques like CUT&RUN or CUT&Tag for validation

• Rescue experiments:

  • Reintroduce wildtype BRD3 or bromodomain mutants

  • Direct targets should be rescued by wildtype but not binding-deficient mutants

• Mechanistic validation:

  • For presumed direct targets, perform detailed analysis of:

    • Transcription factor recruitment

    • RNA polymerase II occupancy

    • Histone modification changes

    • Chromatin accessibility alterations

These approaches help distinguish primary transcriptional effects from secondary responses to BRD3 modulation.

What considerations are important when comparing results across different BRD3 antibodies and experimental systems?

When comparing results from different antibodies or systems:

• Antibody characteristics:

  • Epitope location: Antibodies targeting different regions (N-terminal vs. C-terminal) may detect different isoforms or post-translationally modified forms

  • Clonality: Polyclonal antibodies detect multiple epitopes while monoclonals recognize single epitopes

  • Validation methods: Compare the validation techniques used for each antibody

• Experimental system variables:

  • Cell/tissue types: BRD3 function can be context-dependent across different tissues

  • Species differences: Consider conservation of the BRD3 epitope across species

  • Expression levels: Endogenous vs. overexpression systems yield different results

• Technical parameters:

  • Protocol differences: Variations in fixation, extraction, or detection methods

  • Quantification approaches: Normalization methods and reference genes/proteins

  • Batch effects: Account for experimental variation between batches

• Data reporting standards:

  • Complete antibody information: Catalog numbers, lot numbers, dilutions

  • Detailed methods: Include all protocol parameters for reproducibility

  • Raw data availability: Where possible, provide access to raw data

When conflicting results are observed, consider performing side-by-side comparisons with multiple antibodies under identical conditions to identify the source of variation.

How can BRD3 antibodies be used to study phase separation and biomolecular condensates?

Recent research has revealed that BRD3 undergoes liquid-liquid phase separation (LLPS) upon binding to lncRNA DIGIT , presenting exciting research opportunities:

• Immunofluorescence approaches:

  • Use high-resolution microscopy with BRD3 antibodies to visualize condensate formation

  • Co-staining with RNA markers to verify RNA-dependent condensates

  • Live-cell imaging with fluorescently tagged BRD3 to monitor dynamics

• Biochemical characterization:

  • Differential centrifugation to isolate condensates followed by immunoblotting

  • Turbidity assays with recombinant BRD3 and RNA

  • Analysis of concentration-dependent phase separation behaviors

• Stimulus-response studies:

  • Monitor condensate formation under different cellular stresses

  • Effect of transcriptional inhibitors on BRD3 condensates

  • Role of post-translational modifications in regulating phase separation

• Functional consequences:

  • ChIP-seq following disruption of phase separation

  • Transcriptional outputs of genes regulated by BRD3 condensates

  • Interaction proteomics of proteins recruited to BRD3 condensates

• Therapeutic implications:

  • Screen for compounds that modulate BRD3 phase separation

  • Evaluate correlation between condensate disruption and gene expression

These approaches can address fundamental questions about how BRD3's phase separation properties contribute to its role in promoting binding to acetylated histones and inducing endoderm gene expression .

What are the methodological considerations for studying BRD3's role in inflammatory and stress responses?

Based on recent findings about BRD3's role in regulating inflammatory and stress responses , key methodological approaches include:

• Cell-type specific analyses:

  • Isolation protocols: Standardized methods for obtaining primary cells (e.g., fibroblast-like synoviocytes)

  • Inflammatory stimulation: Defined conditions using TNF-α, IL-1β, or LPS with time course analysis

  • Stress induction protocols: Oxidative stress (H₂O₂), heat shock, or nutrient deprivation

• BRD3 manipulation strategies:

  • Genetic approaches: CRISPR/Cas9 knockout, siRNA/shRNA knockdown

  • Pharmacological approaches: BET inhibitors with varying selectivity profiles

  • Domain-specific mutants: Bromodomain mutants vs. ET domain mutants

• Readout systems:

  • Cytokine/chemokine production: ELISA, multiplex assays, qRT-PCR

  • Stress response markers: Western blotting for p62, LC3B (autophagy markers)

  • Metabolic measurements: Seahorse assays, metabolomics

• Temporal considerations:

  • Acute vs. chronic inflammatory models

  • Resolution phase analysis

  • Adaptation mechanisms under prolonged stress

• Pathway analysis:

  • Dissection of signaling cascades (NF-κB, MAPK, etc.)

  • Intersection with metabolic pathways

  • Integration with stress response networks

These approaches should incorporate appropriate controls including other BET family members (BRD2, BRD4) to distinguish BRD3-specific effects from general BET protein functions .

What are the latest methodological advances for studying BRD3 interactions with non-histone acetylated proteins?

Recent advances for studying BRD3-protein interactions include:

• Proximity-based labeling techniques:

  • BioID or TurboID fusion with BRD3 to identify proximal interacting proteins

  • APEX2-BRD3 fusions for temporal interaction mapping

  • These methods capture transient interactions often missed by traditional co-IP

• Mass spectrometry-based approaches:

  • Acetylome profiling to identify candidate BRD3-interacting proteins

  • SILAC or TMT labeling to quantify differential interactions under various conditions

  • Crosslinking mass spectrometry (XL-MS) to map interaction interfaces

• High-resolution microscopy:

  • Super-resolution imaging to visualize co-localization at sub-diffraction resolution

  • FRET or FLIM to detect direct protein-protein interactions

  • Single-molecule tracking to monitor binding dynamics

• Biochemical interaction mapping:

  • Peptide arrays with acetylated and non-acetylated versions of candidate interactors

  • Surface plasmon resonance or biolayer interferometry for binding kinetics

  • Hydrogen/deuterium exchange mass spectrometry to map binding interfaces

• Functional validation strategies:

  • Acetylation site mutations in partner proteins

  • Bromodomain-specific inhibitors to disrupt specific interactions

  • Domain swapping between BET proteins to identify specificity determinants

These methodologies build upon foundational work showing BRD3's interaction with acetylated GATA1 and GATA2 , enabling more comprehensive characterization of BRD3's non-histone protein interactions.

What key reference studies have established the functional roles of BRD3 in gene regulation?

Seminal studies characterizing BRD3 function include:

• GATA1 interaction and hematopoietic regulation:

  • Lamonica et al. (2011) demonstrated that acetylated GATA1 binds BRD3 in an acetylation-dependent manner to facilitate stable association with chromatin

  • BRD3 was shown to occupy most GATA1-occupied regulatory DNA elements genome-wide

  • This study established the role of BRD3 as a "reader" of non-histone acetylated proteins

• Chromatin association mechanisms:

  • LeRoy et al. (2008) showed that BRD3 binds acetylated lysine residues on histones H2A, H2B, H3 and H4

  • Demonstrated BRD3's role in nucleosome remodeling during transcription

• Phase separation and gene regulation:

  • Daneshvar et al. (2020) revealed that BRD3 undergoes liquid-liquid phase separation upon binding to lncRNA DIGIT

  • This phase separation promotes binding to H3K18ac and induces endoderm gene expression

• Inflammatory and stress responses:

  • Recent work has highlighted BRD3's role in regulating cytokine and chemokine production

  • BRD3 has been implicated in metabolic and stress-related adaptation under inflammatory conditions

• Cancer connections:

  • The BRD3 gene can be fused to NUT in NUT midline carcinomas

  • BET inhibitors competing with BRD3 and BRD4 for chromatin binding can induce apoptosis in MLL-fusion driven leukemic cell lines

These studies collectively establish BRD3 as a multifunctional chromatin reader with roles in transcriptional regulation, cell differentiation, inflammation, and disease processes.