BRD7 Antibody

What is BRD7 and why is it significant in research?

BRD7 (bromodomain containing 7) is a 651 amino acid transcriptional regulator with a calculated molecular weight of approximately 74 kDa that contains a single bromodomain . It functions as a component of chromatin remodeling complexes and is suggested to possess histone acetyltransferase activity . BRD7 plays critical roles in:

• Cell growth and cell cycle progression regulation

• Signal-dependent gene expression

• Tumor suppression by negatively regulating key cell cycle proteins such as cyclin D1 and E2F-3

• Inhibiting G1 to S phase transition in cell cycle progression

Research significance stems from BRD7's involvement in various cancer types, particularly its inhibitory effects on nasopharyngeal carcinoma cell growth and its recently discovered role in metastatic dormancy in breast cancer .

What are the common applications for BRD7 antibodies?

BRD7 antibodies are utilized in multiple experimental techniques across cancer biology, epigenetics, and cell cycle research:

Experimental design should include appropriate controls and antibody validation to ensure specific detection of BRD7 .

How do I select the appropriate BRD7 antibody for my research?

Selection of an appropriate BRD7 antibody depends on multiple experimental factors:

• Target species: Verify reactivity with your experimental model (human, mouse, rat, etc.). Many BRD7 antibodies show cross-reactivity between human, mouse, and rat samples .

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

• Antibody type: Consider whether monoclonal or polyclonal antibodies are more suitable:

  • Monoclonal antibodies (e.g., H-2, B-8): Offer high specificity for a single epitope, providing consistent results

  • Polyclonal antibodies: Recognize multiple epitopes, potentially providing stronger signals but with potential for higher background

• Epitope location: For domain-specific studies, select antibodies raised against relevant regions of BRD7.

• Validation data: Review publications that have successfully used the antibody, focusing on those employing similar experimental conditions to your research .

• Controls: Plan appropriate positive controls (tissues/cells known to express BRD7) and negative controls (knockdown/knockout systems) .

What are the optimal protocols for BRD7 antibody validation?

Rigorous validation ensures experimental reliability and reproducibility:

• Western blot validation:

  • Verify band size corresponds to predicted molecular weight (70-74 kDa)

  • Include positive controls (cells known to express BRD7, such as COLO 320, HeLa, or Jurkat cells)

  • Include negative controls (BRD7 knockdown or knockout samples)

  • Test specificity using recombinant BRD7 protein competition assays

• Immunoprecipitation validation:

  • Confirm ability to immunoprecipitate BRD7 by western blot detection

  • Verify using tag-based detection (e.g., Myc-tagged BRD7) as demonstrated in previous studies

  • Example: BRD7-Myc protein was detected by c-Myc antibody in immunocomplexes precipitated with anti-BRD7 antibody

• Cross-reactivity assessment:

  • Test antibody against closely related proteins (especially BRD9, which shares 73.2% sequence identity with BRD7's bromodomain)

  • Evaluate specificity across multiple cell lines and tissues

• Functional validation:

  • Correlate antibody detection with functional assays (e.g., cell cycle analysis in BRD7 overexpression/knockdown models)

  • Confirm subcellular localization patterns (primarily nuclear for wild-type BRD7)

How can BRD7 antibodies be utilized in cancer research and metastasis studies?

BRD7 antibodies enable investigation of tumor suppressor functions and metastatic mechanisms:

• Tumor suppressor mechanisms:

  • BRD7 negatively regulates cell cycle proteins including cyclin D1 and E2F-3

  • Antibodies can be used to track BRD7 expression changes during carcinogenesis

  • IHC/IF applications reveal expression patterns in various cancer tissues

• Metastasis research:

  • Recent findings show Brd7 loss induces metastatic reawakening in breast cancer models

  • BRD7 antibodies can monitor expression changes during metastatic progression

  • Applications in studying how Brd7 loss modifies epigenomic landscapes and upregulates oncogenic signaling

• Immune microenvironment analysis:

  • BRD7-deficient metastases reprogram surrounding immune environments by downregulating MHC-1 expression and promoting pro-metastatic cytokine profiles

  • Multiplex IF with BRD7 antibodies can reveal spatial relationships with immune cells

  • Flow cytometry applications to study correlations between BRD7 expression and immune cell populations

• Therapeutic target assessment:

  • Evaluating BRD7 expression changes in response to bromodomain inhibitors

  • Monitoring BRD7 expression during neutrophil depletion, NET inhibition, or immune checkpoint therapy, which can abrogate metastatic outgrowth

What are the technical challenges when using BRD7 antibodies for chromatin-related studies?

Investigating BRD7's role in chromatin remodeling complexes presents specific technical considerations:

• Crosslinking efficiency in ChIP experiments:

  • BRD7 functions within large chromatin remodeling complexes (PBAF)

  • Optimization of crosslinking conditions is critical for capturing genuine chromatin interactions

  • Sequential ChIP (ChIP-reChIP) may be necessary to distinguish BRD7-specific binding from other PBAF components

• Epitope accessibility challenges:

  • BRD7's incorporation into chromatin complexes may mask epitopes

  • Testing multiple antibodies recognizing different regions of BRD7 is recommended

  • Epitope retrieval methods require optimization for IHC/IF of chromatin-bound BRD7

• Distinguishing direct vs. indirect chromatin interactions:

  • BRD7 may interact with chromatin directly via its bromodomain or indirectly through protein partners

  • Controls with bromodomain inhibitors can help distinguish these interactions

  • Paired analyses with BRD7 antibodies and histone modification antibodies provide functional context

• Post-translational modification detection:

  • Western blot analysis has shown potential post-translational modifications of endogenously expressed BRD7, appearing as doublet bands

  • Modified BRD7 may have altered antibody recognition properties

  • Consider phospho-specific or other modification-specific antibodies when available

How can I optimize western blot protocols for BRD7 detection?

Optimizing western blot conditions ensures reproducible BRD7 detection:

• Sample preparation considerations:

  • Nuclear extraction protocols are recommended as BRD7 is primarily nuclear

  • Inclusion of phosphatase inhibitors may help preserve post-translational modifications

  • Fresh samples typically yield better results than frozen samples

• Dilution optimization:

  • Start with manufacturer's recommended dilution range (e.g., 1:5000-1:50000)

  • Perform serial dilutions to determine optimal antibody concentration

  • Sample-dependent titration may be necessary for different cell/tissue types

• Expected band patterns:

  • Primary band at 70-74 kDa corresponding to full-length BRD7

  • Potential doublet bands representing post-translational modifications or splice variants

  • Verify specificity with positive controls such as recombinant BRD7 or BRD7-overexpressing cells

• Common troubleshooting steps:

  • Weak signal: Increase antibody concentration, extend incubation time, or improve protein transfer

  • High background: Increase blocking time, use alternative blocking reagents, or increase washing steps

  • No signal: Verify BRD7 expression in your samples using known positive controls (e.g., HeLa cells)

  • Multiple bands: Validate specificity with knockout/knockdown controls; consider splice variants or degradation products

What considerations are important for IHC/IF applications of BRD7 antibodies?

Optimizing immunostaining protocols for BRD7 detection:

• Antigen retrieval optimization:

  • Test both TE buffer pH 9.0 and citrate buffer pH 6.0 for optimal epitope exposure

  • Determine optimal retrieval time and temperature for your specific tissue samples

  • Different fixation methods may require different retrieval approaches

• Expected staining patterns:

  • Predominantly nuclear staining in most cell types

  • Strong nuclear expression in cerebellum, pancreas, intestines, liver, and kidney

  • Cytoplasmic expression has been observed in cardiomyocytes

  • Weak nuclear expression in cerebrum, lung, and stomach

• Dilution optimization:

  • Begin with recommended range (e.g., 1:20-1:200 for IHC)

  • Titrate antibody concentration using known positive controls

  • Tissue-specific optimization may be required

• Background reduction strategies:

  • Extend blocking time using appropriate blocking reagents

  • Include additional washing steps

  • Consider using species-specific secondary antibodies with minimal cross-reactivity

  • Test signal amplification systems for tissues with low BRD7 expression

How do I interpret different BRD7 expression patterns across tissues and cell types?

Tissue and cell-type specific BRD7 expression patterns provide important biological insights:

• Normal tissue expression profiles:

  • High nuclear expression: cerebellum, pancreas, intestines, liver, kidney

  • High cytoplasmic expression: cardiomyocytes

  • Moderate nuclear expression: liver, intestines, kidney

  • Weak nuclear expression: cerebrum, lung, spleen, stomach

• Cell line expression variations:

  • Positive expression detected in: COLO 320 cells, HeLa cells, Jurkat cells, MDA-MB-231 cells, PC-3 cells, SH-SY5Y cells, L02 cells

  • Expression levels may vary significantly between cell lines

  • Consider tissue of origin when selecting appropriate experimental models

• Subcellular localization significance:

  • Predominantly nuclear localization correlates with transcriptional regulatory functions

  • Cytoplasmic expression in cardiomyocytes suggests potential tissue-specific functions

  • Altered localization may indicate functional changes in disease states

• Developmental considerations:

  • BRD7 expression has been documented in fetal tissues, suggesting roles in development

  • Expression patterns may change during differentiation, similar to other bromodomain proteins like BRD1

What are the considerations for analyzing BRD7 in the context of metastasis and cancer progression?

Interpreting BRD7 experimental results in cancer and metastasis studies:

• Expression changes during cancer progression:

  • Decreased BRD7 expression may correlate with metastatic potential

  • Loss of BRD7 can induce metastatic reawakening in dormant cancer cells

  • Changes in BRD7 expression should be correlated with clinical outcomes data when available

• Epigenomic landscape analysis:

  • BRD7 loss modifies epigenomic landscapes and upregulates oncogenic signaling

  • ChIP-seq with BRD7 antibodies can map altered binding sites during cancer progression

  • Integration with histone modification data provides functional context

• Immune microenvironment considerations:

  • BRD7 loss correlates with downregulated MHC-1 expression and pro-metastatic cytokine profiles

  • Flow cytometric data may show increased levels of:

    • Pro-tumorigenic inflammatory neutrophils

    • Transitional neutrophils

    • CD8+ exhausted T cells

    • CD4+ stress response T cells

  • Consider multiplex analysis with immune cell markers to understand spatial relationships

• Therapeutic response markers:

  • Changes in BRD7 expression may predict response to:

    • Neutrophil depletion therapy

    • Neutrophil extracellular trap (NET) inhibition

    • Immune checkpoint therapy

  • Validation of BRD7 as a biomarker requires correlation with treatment outcomes

How might BRD7 antibodies contribute to emerging research in cancer immunotherapy?

Recent discoveries position BRD7 at the intersection of epigenetic regulation and anti-tumor immunity:

• Immune checkpoint therapy connections:

  • BRD7 loss promotes immunosuppressive microenvironments in metastatic settings

  • BRD7 antibodies can help stratify patients who might benefit from immune checkpoint inhibitors

  • Monitoring BRD7 expression during therapy may predict response or resistance mechanisms

• Neutrophil-targeting approaches:

  • BRD7 deficiency correlates with increased pro-tumorigenic inflammatory neutrophils

  • Combination studies of BRD7 status and neutrophil-targeting therapies require BRD7 antibodies for patient stratification

  • Potential development of companion diagnostics using BRD7 antibodies

• MHC-I downregulation mechanisms:

  • BRD7 loss leads to downregulation of MHC-I expression

  • BRD7 antibodies can help elucidate the epigenetic mechanisms controlling MHC-I expression

  • Development of strategies to reverse this immune evasion mechanism

• Biomarker development:

  • Multiplex IHC panels including BRD7 and immune cell markers

  • Development of quantitative assays for monitoring BRD7 expression during treatment

  • Potential liquid biopsy applications if BRD7 or its downstream targets are detectable in circulation

What are emerging techniques for studying BRD7 using specialized antibody applications?

Advanced methodologies for investigating BRD7's functions:

• Proximity ligation assays (PLA):

  • Detecting in situ protein-protein interactions between BRD7 and other PBAF components

  • Visualizing spatial relationships between BRD7 and transcription factors or chromatin marks

  • Requires highly specific antibodies from different host species

• CUT&RUN and CUT&Tag applications:

  • Higher resolution alternatives to conventional ChIP for mapping BRD7 genomic binding sites

  • Lower background and input requirements than traditional ChIP

  • Requires optimization of antibody binding under native conditions

• Single-cell approaches:

  • Single-cell western blot for heterogeneity analysis of BRD7 expression

  • Mass cytometry (CyTOF) with metal-conjugated BRD7 antibodies

  • Integration with single-cell RNA-seq to correlate BRD7 protein levels with transcriptional changes

• Live-cell imaging:

  • Development of intrabodies or labeled nanobodies against BRD7

  • Monitoring BRD7 dynamics during cell cycle progression and cancer drug responses

  • FRET-based approaches to study BRD7 interactions in living cells