BRD8 Antibody

What is BRD8 and why is it important in epigenetic research?

BRD8 (bromodomain containing 8) is a critical bromodomain-containing protein that functions as an acetylated lysine-binding domain involved in the regulation of protein acetylation and histone acetyl transferase (HAT) activity . It serves as an accessory subunit of the NuA4 HAT complex (also known as TRRAP/TIP60 complex) and has been biochemically identified through purification studies . Additionally, BRD8 can bind to thyroid hormone receptor-β and retinoid X receptor, suggesting its involvement in hormone-mediated transcriptional regulation. Recent research has revealed BRD8's function as an epigenetic rheostat that maintains cell fates, with its depletion potentially disrupting established cellular states . Understanding BRD8 is essential for researchers investigating epigenetic mechanisms, transcriptional regulation, and cellular differentiation processes.

What applications are BRD8 antibodies validated for in research settings?

BRD8 antibodies have been validated for multiple experimental applications across different research settings. The primary applications include:

Researchers should note that optimal dilutions may be sample-dependent, and it is recommended to titrate the antibody in each specific testing system to obtain optimal results . When designing experiments, consider that different antibodies target various epitopes (e.g., middle region, N-terminal domain, specific amino acid ranges) and may perform differently depending on experimental conditions .

What species reactivity should be considered when selecting a BRD8 antibody?

When selecting a BRD8 antibody for research, cross-species reactivity is an important consideration that impacts experimental design and interpretation. Based on available antibody characterization data:

How does the bromodomain of BRD8 differ from other bromodomain-containing proteins in terms of inhibitor sensitivity?

The bromodomain of BRD8 exhibits distinctive characteristics that differentiate it from other bromodomain-containing proteins, particularly regarding inhibitor sensitivity. Unlike the bromodomain of BRD4, which is sensitive to JQ1 (a traditional pan-BET bromodomain inhibitor), BRD8's interaction with histone H4 is not affected by JQ1 treatment . This pharmacological distinction is supported by phylogenetic analysis that positions BRD8 bromodomains in a separate branch from other bromodomains in the evolutionary tree .

The molecular basis for this difference lies in the unique structural features of BRD8's bromodomain. Research has demonstrated that BRD8's bromodomain is not only essential for its interaction with histone H4 but also contains characteristic amino acid sequences required for specific protein-protein interactions, such as with MRGBP (MRG domain binding protein) . This interaction is critical for BRD8 stability, as deletion mutants lacking the bromodomain showed markedly reduced protein stability.

For researchers investigating bromodomain inhibitors or designing targeted therapies, these functional differences highlight the importance of developing BRD8-specific approaches rather than relying on pan-bromodomain compounds. Experimental designs should consider these distinctions when studying BRD8's functional roles or when using BRD8 as a comparison point for other bromodomain proteins.

What are the methodological considerations for detecting BRD8 in different cellular compartments using immunofluorescence?

When designing immunofluorescence experiments to detect BRD8 in different cellular compartments, several methodological considerations must be addressed:

• Fixation method selection: BRD8 detection requires careful consideration of fixation protocols that preserve its native confirmation while enabling antibody access. Paraformaldehyde fixation (4%) for 10-15 minutes is generally recommended, though optimization may be necessary for specific cell types.

• Permeabilization protocol: Since BRD8 has both nuclear and potential cytoplasmic functions, optimization of permeabilization conditions is critical. Triton X-100 (0.1-0.5%) is commonly used, but gentler detergents like saponin may be preferred when studying potential cytoplasmic fractions.

• Validated cell systems: Positive immunofluorescence has been confirmed in HepG2 cells and HEK-293 cells at dilutions of 1:1000-1:4000 . These systems serve as appropriate positive controls when establishing new detection protocols.

• Co-localization markers: For accurate subcellular localization, co-staining with compartment-specific markers is recommended. Nuclear co-localization with DAPI is expected for BRD8's primary function, but potential association with specific chromatin regions may require co-staining with histone modification markers (H3K27ac, H4K5ac, etc.).

• Signal amplification: For detecting potentially low expression levels in certain cell types, signal amplification systems (tyramide signal amplification or quantum dots) may be necessary when standard detection methods yield insufficient signal-to-noise ratios.

Researchers should also consider employing super-resolution microscopy techniques for more detailed subcellular localization studies, particularly when investigating BRD8's dynamic association with chromatin during cell cycle progression or differentiation events.

How does BRD8 function independently of the TIP60 complex, and what experimental approaches can distinguish these roles?

BRD8 has been discovered to function both as part of the NuA4/TIP60-histone acetyltransferase complex and independently through distinct mechanisms. Transcriptome analysis coupled with genome-wide mapping of BRD8-binding sites has revealed that BRD8 can transactivate sets of genes independently of TIP60 . This dual functionality presents significant challenges for researchers attempting to distinguish between TIP60-dependent and TIP60-independent roles.

To experimentally distinguish these functions, researchers can employ several strategic approaches:

• Comparative ChIP-seq analysis: Performing parallel ChIP-seq for BRD8 and TIP60 can identify genomic regions where only BRD8 binds without TIP60 co-occupancy. These regions likely represent TIP60-independent functions. This approach revealed BRD8's independent regulation of pre-replicative complex subunits in concert with the activator protein-1 .

• Sequential immunoprecipitation: Utilizing tandem immunoprecipitation with BRD8 antibodies followed by separation of TIP60-associated and TIP60-free fractions can help isolate protein complexes specific to each functional mode.

• Domain-specific mutants: Generating domain-specific BRD8 mutants that selectively disrupt TIP60 interaction while preserving other functions can help delineate pathway-specific effects. Research has shown that the bromodomain of BRD8 is critical not only for histone interaction but also for protein stability and specific protein-protein interactions .

• Conditional knockdown with rescue experiments: Depleting endogenous BRD8 followed by rescue with wild-type or TIP60-interaction deficient mutants can reveal phenotypes specifically associated with TIP60-independent functions.

• Transcriptome analysis with pathway inhibition: Coupling RNA-seq with selective inhibition of TIP60 acetyltransferase activity can distinguish gene expression changes dependent on BRD8-TIP60 cooperation versus BRD8-autonomous regulation.

These approaches have revealed that BRD8's TIP60-independent functions include regulation of cell cycle progression through the pre-replicative complex and influence on cellular differentiation states .

What are the optimal conditions for antigen retrieval when using BRD8 antibodies for immunohistochemistry?

Successful immunohistochemical detection of BRD8 requires careful optimization of antigen retrieval conditions to expose epitopes that may be masked during fixation processes. Based on validated protocols, the following antigen retrieval approaches have proven effective:

Primary recommendation: TE buffer (10 mM Tris, 1 mM EDTA) at pH 9.0 has been validated for optimal BRD8 epitope exposure in multiple tissue types, particularly in human colon cancer tissue . This alkaline buffer system effectively breaks protein cross-links formed during formalin fixation without excessive tissue damage.

When troubleshooting suboptimal IHC results, consider these methodological modifications:

• Heating method variation: Compare water bath, microwave, and pressure cooker-based heating for 15-20 minutes to determine optimal epitope recovery for your specific tissue type.

• Incubation time adjustment: For highly fixed tissues (>24h in fixative), extending the retrieval time to 25-30 minutes may improve signal intensity.

• Enzymatic pre-treatment: For particularly challenging samples, a brief (5-10 minute) enzymatic pre-treatment with proteinase K (10 μg/ml) prior to heat-induced epitope retrieval may enhance antibody accessibility.

• Post-retrieval cooling: Allow slides to cool gradually in the retrieval solution for 20-30 minutes before proceeding to blocking steps to prevent tissue detachment and reduce background.

• Buffer additives: Addition of 0.05% Tween-20 to retrieval buffers can improve penetration of retrieval solution into the tissue.

Optimization should be performed on control tissues with known BRD8 expression, and conditions should be standardized across experimental cohorts to ensure consistent detection sensitivity and comparability of results.

How can researchers validate antibody specificity for BRD8 and avoid cross-reactivity issues?

Validating antibody specificity for BRD8 is critical for generating reliable and reproducible research data. A comprehensive validation strategy should include:

• CRISPR/siRNA validation: Utilize BRD8 knockdown or knockout models as negative controls. Western blot or immunostaining should show significant reduction or absence of signal at the expected molecular weight (120 kDa) . This approach provides the most definitive evidence of specificity.

• Recombinant protein controls: Test antibody recognition of purified recombinant BRD8 protein or fragments containing the target epitope. Blocking experiments with immunizing peptides can confirm epitope-specific binding.

• Multi-antibody verification: Compare detection patterns using antibodies targeting different BRD8 regions (N-terminal, middle region, C-terminal) . Consistent detection patterns across multiple antibodies increases confidence in specificity.

• Known expression pattern correlation: Compare your findings with published BRD8 expression datasets from RNA-seq or proteomics studies. Tissue or cell-type expression patterns should generally correlate with transcript levels.

• Mass spectrometry verification: For definitive validation, immunoprecipitate BRD8 with your antibody and confirm protein identity using mass spectrometry analysis of the precipitated proteins.

To avoid cross-reactivity issues, especially with other bromodomain-containing proteins:

• Select antibodies raised against unique regions of BRD8 that have limited sequence homology with other bromodomain proteins

• Use appropriate blocking with 3-5% BSA or 5% normal serum from the same species as your secondary antibody

• Include additional controls when working with tissues expressing multiple bromodomain proteins

• Consider pre-absorption of antibodies with related proteins when cross-reactivity is suspected

For Western blot applications, detecting BRD8 at its expected molecular weight of 120 kDa provides additional confidence in specificity .

How does BRD8 contribute to cell cycle regulation in cancer cells, and what techniques best capture this dynamic process?

BRD8 plays a critical role in cell cycle regulation in cancer cells, particularly through its regulation of pre-replicative complex (pre-RC) components. Research in colorectal cancer cells has revealed that BRD8 transactivates multiple subunits of the pre-RC complex in concert with activator protein-1 (AP-1) . Depletion of BRD8 induces cell cycle arrest at the G1 phase and suppresses cell proliferation, indicating its essential role in cell cycle progression .

To effectively study BRD8's dynamic role in cell cycle regulation, researchers should consider these specialized techniques:

• Cell synchronization with time-course analysis: Synchronizing cells at specific cell cycle stages (e.g., double thymidine block, nocodazole arrest) followed by BRD8 ChIP-seq at defined time points can map temporal changes in BRD8 genomic occupancy throughout the cell cycle.

• Proximity ligation assays (PLA): This technique can visualize and quantify interactions between BRD8 and cell cycle regulatory proteins in situ, providing spatial and temporal resolution of complex formation during cell cycle progression.

• FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator) system integration: Combining BRD8 immunofluorescence with FUCCI cell cycle reporters allows precise correlation of BRD8 localization and activity with specific cell cycle phases at the single-cell level.

• Live-cell imaging with fluorescently tagged BRD8: Generation of cell lines expressing fluorescently tagged BRD8 enables real-time visualization of its dynamics during cell division and in response to cell cycle perturbations.

• Sequential ChIP (Re-ChIP): This approach can distinguish genomic loci where BRD8 and AP-1 co-occupy pre-RC gene promoters versus independent binding sites, clarifying cooperative transcriptional mechanisms.

• Nascent RNA labeling: Techniques like BRIC-seq or EU-seq combined with BRD8 manipulation can directly measure the impact of BRD8 on transcription rates of cell cycle-related genes rather than steady-state mRNA levels.

Recent findings on BRD8's role in colorectal cancer suggest that it accumulates in cancer cells through inhibition of ubiquitin-dependent protein degradation, mediated by interaction with MRG domain binding protein . This stabilization mechanism may contribute to the maintenance of a proliferative phenotype, suggesting that disrupting BRD8 stability could represent a therapeutic approach for cancers with elevated BRD8 levels.

What is the role of BRD8 in cellular differentiation and stem cell maintenance, and how can researchers investigate these functions?

BRD8 functions as an epigenetic rheostat that maintains cell fate decisions and influences cellular differentiation trajectories. Recent research has revealed that BRD8 guards the pluripotent state by sensing and maintaining specific epigenetic modifications . Reduced BRD8 levels have been shown to promote the transition of epiblast stem cells (EpiSCs) to embryonic stem cells (ESCs) in primed-to-naïve conversion processes .

To investigate BRD8's role in cellular differentiation and stem cell maintenance, researchers can employ these specialized approaches:

Research has demonstrated that BRD8 binds to naïve-specific gene loci and modulates epigenetic modifications at those sites, thereby influencing cell type transitions . This process requires the action of histone deacetylases and a catalytically active histone acetyltransferase KAT5 (TIP60), suggesting a complex interplay between epigenetic modifiers in maintaining cellular identity .

What are the optimal storage and handling conditions for maintaining BRD8 antibody activity?

Proper storage and handling of BRD8 antibodies is critical for maintaining their activity and ensuring consistent experimental results. Based on manufacturer recommendations and best practices:

Storage conditions:

• Store BRD8 antibodies at -20°C for long-term preservation

• Antibodies formulated with 50% glycerol and PBS with 0.02% sodium azide at pH 7.3 remain stable for one year after shipment

• Aliquoting is not necessary for -20°C storage of glycerol-formulated antibodies, but is recommended for antibodies without cryoprotectants

• Avoid repeated freeze-thaw cycles (more than 5) which can lead to antibody degradation and reduced sensitivity

Working solution preparation:

• When preparing working dilutions, use fresh, cold buffer (PBS or TBS with 0.1% BSA)

• For Western blot applications, prepare dilutions in 5% non-fat milk or BSA in TBST immediately before use

• For immunofluorescence applications, dilute in 1% BSA in PBS with 0.1% Tween-20

• Working solutions should ideally be used within 24 hours and not stored for extended periods

Handling precautions:

• Small volume antibodies (20μl sizes) often contain 0.1% BSA as a stabilizer

• For sensitive applications like immunofluorescence, centrifuge the antibody briefly before opening to collect liquid at the bottom of the vial

• Always use clean, DNase/RNase-free pipette tips when handling antibody stocks

• Record the number of freeze-thaw cycles on each vial to monitor potential activity loss

Stability testing:

• If stored for extended periods (>6 months), perform validation tests on known positive controls before crucial experiments

• For antibodies approaching expiration dates, comparative titration against a new lot is recommended to assess potential sensitivity loss

Proper storage and handling not only preserves antibody activity but also enhances experimental reproducibility and reduces the variability that can complicate data interpretation across experiments.

How can researchers optimize western blot conditions for detecting BRD8 in different tissue and cell types?

Optimizing western blot conditions for BRD8 detection requires careful consideration of sample preparation, electrophoresis, and detection parameters, particularly given BRD8's relatively high molecular weight (120 kDa) . For successful detection across diverse tissue and cell types, consider these technical optimizations:

Sample preparation:

• Use RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors for efficient extraction

• For nuclear-enriched fractions (recommended for BRD8), employ gentle nuclear lysis buffers (0.1% NP-40 followed by high-salt nuclear extraction)

• Sonicate samples briefly (3-5 pulses) to shear genomic DNA and improve protein release

• Heat samples at 70°C instead of 95-100°C to prevent aggregation of high molecular weight proteins

Gel electrophoresis optimization:

• Use lower percentage gels (6-8% acrylamide) or gradient gels (4-15%) to improve resolution in the high molecular weight range

• Extend running time at lower voltage (80-100V) to ensure complete separation

• Include molecular weight markers that clearly delineate the 100-130 kDa range

Transfer conditions:

• For proteins >100 kDa, use wet transfer systems rather than semi-dry methods

• Transfer at 30V overnight at 4°C for improved efficiency of large proteins

• Use 0.45 μm PVDF membranes rather than 0.2 μm for better retention of high MW proteins

• Add 0.1% SDS to transfer buffer to facilitate movement of large proteins

Detection optimization:

• Validated dilution range for Western blot: 1:500-1:2000 (start with 1:1000 and adjust based on signal strength)

• Extend primary antibody incubation to overnight at 4°C for improved sensitivity

• Use high-sensitivity ECL substrates for detecting potentially low abundance BRD8 in certain tissues

• Consider signal amplification systems for tissues with naturally low BRD8 expression

Tissue-specific considerations:

• Fetal human brain tissue and HeLa cells have been validated as positive controls

• For tissues with high lipid content (brain, adipose), additional sample clarification steps may be necessary

• For muscle tissues, extended lysis times and mechanical disruption improve protein extraction

Researchers should systematically titrate antibody concentrations and optimize blocking conditions (3-5% BSA often performs better than milk for phosphorylated proteins) when working with new tissue types or cell lines to achieve optimal signal-to-noise ratios.