BRE Antibody

What is BRE protein and what cellular functions does it regulate?

BRE (Brain and reproductive organ-expressed protein) is a 415 amino acid protein that functions as a critical modulator of cellular processes. It binds to the intracellular juxtamembrane domain of death receptors, particularly tumor necrosis factor receptor 1 (TNF-R1) and FAS . BRE plays significant roles in downregulating TNFα-induced activation of NFκB and inhibiting components of death-inducing signaling complexes necessary for mitochondrial-mediated apoptosis . The protein exhibits strong expression in adrenal cortex, medulla, testis, and pancreas, with weaker expression in thymus, thyroid, stomach, and small intestine . BRE is particularly important for homeostasis and cellular differentiation in cells of epithelial, neural, and germ line origins, making it a valuable target for studying tissue-specific regulatory mechanisms .

How are BRE antibodies classified and what are their primary research applications?

BRE antibodies are available in multiple formats, primarily as monoclonal and polyclonal variants derived from rabbit hosts. The main classifications include:

These antibodies are predominantly used in cancer research, apoptosis studies, and investigations of cellular stress responses . The monoclonal antibodies provide excellent specificity for quantitative applications like Western blotting, while polyclonal variants offer advantages in detecting native protein conformations in immunohistochemistry applications .

What is the molecular weight of BRE protein and how does this impact antibody selection?

BRE protein has a molecular weight of approximately 45 kDa, as identified through Western blotting applications . When selecting antibodies for BRE detection, researchers should verify that their chosen antibody recognizes this molecular weight in their experimental system. The molecular weight information is critical for:

• Confirming target specificity when visualizing Western blot results

• Distinguishing between potential isoforms or degradation products

• Selecting appropriate gel concentration for optimal separation

• Determining whether post-translational modifications might be affecting migration patterns

Always check the product datasheet for expected molecular weight information and validation data showing the antibody's performance in systems similar to your experimental model .

What validation steps should I perform before using a BRE antibody in critical experiments?

Before employing a BRE antibody in pivotal experiments, comprehensive validation is essential to ensure reliable and reproducible results:

• Validate with known positive and negative controls:

  • Use tissues with documented high BRE expression (adrenal cortex, testis, pancreas) as positive controls

  • Include tissues with minimal expression (or knockout models if available) as negative controls

• Perform technical validation:

  • Determine optimal antibody concentration through titration experiments

  • Verify specificity through pre-absorption with immunizing peptide

  • Consider CRISPR/Cas knockout cell lines for definitive validation

• Cross-reference with literature:

  • Check published studies that have used the same antibody clone

  • Verify the antibody's Research Resource Identifier (RRID) for consistency across studies

• Application-specific validation:

  • For Western blotting: Confirm single band at expected molecular weight (45 kDa)

  • For IHC: Verify expected subcellular localization and expression pattern

  • For IP: Confirm enrichment of target protein after pulldown

This multi-step validation approach, while time-consuming, is crucial for ensuring that subsequent experimental data are built on a foundation of reagent reliability .

How should I optimize sample preparation for detecting BRE protein in different tissues?

Optimal sample preparation is critical for detecting BRE protein accurately across different tissue types:

• For high-expressing tissues (adrenal, testis, pancreas):

  • Use gentler lysis conditions to prevent protein degradation

  • Consider lower antibody concentrations (1:1500-1:2000) to prevent oversaturation

• For low-expressing tissues (thymus, thyroid, stomach):

  • Employ more stringent extraction methods to maximize protein yield

  • Consider sample enrichment techniques like immunoprecipitation before analysis

  • Use higher antibody concentrations (1:500-1:750) to enhance detection sensitivity

• General tissue preparation guidelines:

  • Include protease inhibitors to prevent degradation of BRE protein

  • Optimize homogenization methods based on tissue firmness

  • Consider subcellular fractionation when studying compartment-specific BRE localization

• Sample storage considerations:

  • Limit freeze-thaw cycles to prevent protein degradation

  • Store samples at -80°C for long-term preservation

Tissue-specific optimization is particularly important when comparing BRE expression across different organ systems or when examining disease states that may alter tissue composition .

What controls are essential when using BRE antibodies in cancer research applications?

This comprehensive control strategy is particularly important in cancer research where BRE expression has been linked to patient survival in breast cancer and may predict favorable outcomes in acute myeloid leukemia (AML) . Additionally, BRE overexpression in hepatocellular carcinoma has been associated with resistance to apoptotic signaling and enhanced tumor growth, highlighting the importance of precise quantification .

What are common technical challenges when using BRE antibodies in Western blotting?

Researchers frequently encounter specific challenges when detecting BRE protein via Western blotting:

• Background issues:

  • Problem: High background obscuring specific BRE signal

  • Solution: Increase blocking time/concentration, optimize antibody dilution (start with 1:1000), use higher quality blocking agents

• Multiple bands:

  • Problem: Detection of bands beyond the expected 45 kDa

  • Solution: Confirm specificity with knockout controls, optimize sample preparation to reduce proteolysis, adjust exposure time

• Weak or no signal:

  • Problem: Insufficient detection of BRE despite proper technique

  • Solution: Verify expression in chosen sample type, reduce wash stringency, increase antibody concentration or incubation time, check for interfering post-translational modifications

• Inconsistent results between experiments:

  • Problem: Variable band intensity between replicate experiments

  • Solution: Standardize lysate preparation, avoid repeated freeze-thaw cycles, use freshly prepared buffers, consider recombinant antibodies for improved lot-to-lot consistency

For optimal results with BRE detection, the recommended dilution for Western blotting is 1:1000, though this may require optimization based on your specific experimental system .

How do I optimize BRE antibody protocols for immunohistochemistry applications?

Optimizing BRE antibody protocols for immunohistochemistry requires consideration of several technical parameters:

• Fixation optimization:

  • For formalin-fixed paraffin-embedded tissues: Standardize fixation time (8-24 hours)

  • For frozen sections: Use fresh 4% paraformaldehyde with controlled fixation times

• Antigen retrieval methods:

  • Heat-induced epitope retrieval: Test both citrate (pH 6.0) and EDTA (pH 9.0) buffers

  • Enzymatic retrieval: Consider light proteinase K treatment if heat methods are insufficient

• Blocking optimization:

  • Use serum from the same species as the secondary antibody

  • Add 0.1-0.3% Triton X-100 for improved penetration

  • Consider dual blocking with serum and BSA for reduced background

• Antibody incubation parameters:

  • Test both room temperature (2-3 hours) and 4°C overnight incubation

  • Optimize antibody concentration through serial dilution testing

  • Consider using antibody diluents containing background-reducing components

• Signal development:

  • For brightfield IHC: Compare DAB, AEC, and other chromogens for optimal signal-to-noise

  • For fluorescence: Select fluorophores with minimal spectral overlap with tissue autofluorescence

Systematically testing these variables will help establish the optimal protocol for consistent BRE visualization in tissue sections .

How should I select appropriate housekeeping controls when quantifying BRE expression?

Selection of appropriate housekeeping controls is critical for accurate quantification of BRE expression, especially in disease models where common housekeeping proteins may be regulated:

• Context-specific considerations:

  • For studies involving fibrosis or cellular composition changes: Total protein normalization is preferable to single housekeeping proteins

  • For cancer studies: Verify stability of housekeeping protein expression in your specific cancer model

• Recommended approaches:

  • Use multiple housekeeping controls rather than relying on a single reference

  • Validate stability of housekeeping proteins in your experimental conditions before proceeding

  • Consider stain-free technology or Ponceau staining for total protein normalization

• Avoiding pitfalls:

  • Do not use GAPDH for normalization in tissues with altered metabolic activity

  • Be cautious with β-actin in tissues undergoing structural remodeling

  • Avoid housekeeping proteins in the same molecular weight range as BRE (45 kDa)

For accurate quantification, the practice of using one blot for sequential staining of several different antibodies by stripping and reprobing is discouraged, as each antibody has its own dynamic range of signal intensity .

How does BRE expression correlate with cancer progression and patient outcomes?

BRE expression demonstrates complex relationships with cancer progression and patient outcomes that vary by cancer type:

• Breast cancer:

  • BRE expression levels correlate with patient survival rates

  • Higher expression levels may be associated with better clinical outcomes in certain subtypes

• Acute myeloid leukemia (AML):

  • BRE may serve as a predictive biomarker for favorable outcomes

  • Expression patterns could potentially stratify patients for treatment decisions

• Hepatocellular carcinoma:

  • BRE is frequently overexpressed compared to normal liver tissue

  • Overexpression is associated with resistance to apoptotic signaling

  • May contribute to enhanced tumor growth through anti-apoptotic mechanisms

When designing studies to investigate these correlations, researchers should carefully consider:

• The need for adequate patient sample sizes to achieve statistical power

• Controlling for confounding factors such as treatment history

• Incorporation of multiple detection methods beyond antibody-based approaches

• Integration with genomic and transcriptomic data for comprehensive analysis

This complex relationship between BRE expression and cancer outcomes highlights the importance of context-specific analysis in different cancer types .

How can I distinguish between different BRE protein complexes in cellular systems?

BRE participates in multiple protein complexes with distinct functions, requiring specialized experimental approaches to distinguish between them:

• BRCA1-A complex involvement:

  • BRE functions as BRCA1-A complex subunit BRE (also known as BRCC45)

  • To specifically study this complex:

    • Use co-immunoprecipitation with BRCA1 or other complex components

    • Employ proximity ligation assays to visualize interactions in situ

    • Consider size-exclusion chromatography to separate intact complexes

• Death receptor signaling complexes:

  • BRE interacts with TNF-R1 and FAS

  • To differentiate these interactions:

    • Use stimulus-specific activation (TNFα vs. FasL)

    • Employ sequential immunoprecipitation approaches

    • Consider FRET-based interaction studies for real-time analysis

• General approaches for complex distinction:

  • Blue-native PAGE can separate intact protein complexes

  • Cross-linking mass spectrometry can identify interaction interfaces

  • Sucrose gradient ultracentrifugation can separate complexes by size/density

Distinguishing between these complexes is crucial for understanding BRE's multifaceted roles in cellular homeostasis, DNA damage response, and apoptosis regulation .

What are the key considerations when studying BRE in relation to DNA damage response pathways?

BRE's involvement in DNA damage response pathways presents unique experimental considerations:

• Experimental induction of DNA damage:

  • Select DNA damaging agents based on the pathway of interest:

    • Ionizing radiation for double-strand breaks

    • UV radiation for nucleotide excision repair pathways

    • Replication inhibitors for replication stress response

  • Use dose-response and time-course analyses to capture dynamic changes in BRE localization and complex formation

• Visualization approaches:

  • Immunofluorescence co-localization with γH2AX or 53BP1 to identify BRE recruitment to damage sites

  • Live-cell imaging with fluorescently-tagged BRE to monitor real-time recruitment kinetics

  • Super-resolution microscopy to resolve sub-nuclear structures at damage sites

• Functional assessment methods:

  • BRE knockdown/knockout followed by survival assays after DNA damage

  • Reporter assays to measure repair efficiency in BRE-deficient cells

  • Chromatin immunoprecipitation to assess BRE recruitment to specific genomic loci after damage

• Interaction studies:

  • Investigate BRE's relationship with the BRCA1-A complex components

  • Assess how DNA damage affects BRE's association with different protein partners

  • Determine how post-translational modifications regulate these interactions

These approaches can help elucidate BRE's contributions to genome stability maintenance and DNA repair processes, which may have implications for cancer therapy resistance mechanisms .

How should I interpret contradictory results between different BRE antibody clones?

When faced with contradictory results between different BRE antibody clones, a systematic troubleshooting approach is essential:

• Epitope mapping analysis:

  • Different antibodies may recognize distinct epitopes that are differentially accessible in certain conditions

  • Determine the immunizing peptide/region for each antibody

  • Consider whether post-translational modifications might affect epitope recognition

• Validation status comparison:

  • Evaluate the validation evidence for each antibody

  • Prioritize results from antibodies with stronger validation credentials

  • Consider conducting knockout/knockdown controls for each antibody

• Technical reconciliation approaches:

  • Test both antibodies under identical conditions

  • Employ orthogonal methods (mRNA analysis, mass spectrometry) to resolve contradictions

  • Consider using antibody combinations targeting different epitopes

• Documentation and reporting recommendations:

  • Transparently report contradictory findings in publications

  • Provide complete antibody identification information (catalog number, clone, lot)

  • Discuss potential biological explanations for discrepancies

This systematic approach helps distinguish between technical artifacts and genuine biological complexity when interpreting contradictory BRE antibody results .

What strategies can improve reproducibility when working with BRE antibodies across different laboratories?

Enhancing reproducibility of BRE antibody experiments across laboratories requires systematic approaches:

• Standardized reporting practices:

  • Document complete antibody information: manufacturer, catalog number, lot number, clone ID, and RRID (Research Resource Identifier)

  • Specify exact experimental conditions: dilutions, incubation times/temperatures, detection methods

  • Share original unprocessed blot images with molecular weight markers visible

• Validation sharing:

  • Include validation data in publications and supplementary materials

  • Deposit validation protocols in repositories like Protocols.io

  • Reference antibody validation resources from Table 1 in the literature

• Control implementation:

  • Consistently use positive and negative controls as outlined in Table 2

  • Include biological reference materials when possible

  • Implement blinded analysis procedures for subjective assessments

• Methodology standardization:

  • Develop and share detailed standard operating procedures

  • Specify exact buffer compositions rather than generic descriptions

  • Adopt field-wide standards for common procedures

These practices directly address the "reproducibility crisis" in antibody-based research by providing sufficient methodological transparency for replication efforts .

How can BRE antibody studies be integrated with other multi-omics approaches for comprehensive research?

Integrating BRE antibody studies with multi-omics approaches creates opportunities for deeper biological insights:

• Transcriptomics integration:

  • Correlate protein-level BRE detection with mRNA expression data

  • Identify potential post-transcriptional regulation mechanisms

  • Use RNA-seq to identify co-regulated gene networks

• Proteomics complementation:

  • Validate antibody findings with mass spectrometry-based quantification

  • Use IP-MS to identify novel BRE interaction partners

  • Employ phospho-proteomics to map BRE-regulated signaling networks

• Functional genomics approaches:

  • Combine CRISPR screens with BRE antibody-based phenotyping

  • Identify genetic dependencies that influence BRE expression or function

  • Use ChIP-seq to map transcriptional regulation of BRE

• Clinical data integration:

  • Correlate BRE immunohistochemistry results with patient outcomes

  • Develop integrated biomarker panels combining BRE with other markers

  • Build predictive models incorporating multiple data types

This integrated approach provides a systems-level understanding of BRE biology that extends beyond what antibody-based studies alone can achieve, potentially revealing new therapeutic targets or biomarker applications .