BRAP Antibody

What is BRAP and why is it significant in research?

BRAP (Bombesin Receptor-Activated Protein), also known as BRCA1-associated protein, RNF52, BRAP2, or IMP, is a 592 amino acid cytoplasmic protein featuring UBP-type and RING-type zinc fingers. It functions as a Ras-responsive E3 ubiquitin ligase and plays crucial roles in cellular signaling by negatively regulating MAP kinase activity through Ksr-1 scaffold protein inactivation. This regulation is vital for controlling cell proliferation and differentiation, making BRAP significant in cancer biology, pulmonary inflammation, liver morphology, and cilia development .

What are the structural characteristics of BRAP protein?

BRAP is a 67 kDa protein (predicted molecular weight) that typically appears as two bands of similar molecular weight at ~65 kDa in Western blot analyses. It contains one UBP-type zinc finger and one RING-type zinc finger domain. The protein is primarily localized in the cytoplasm and undergoes auto-ubiquitination as part of its function as an E3 ubiquitin ligase. The BRAP gene is located on human chromosome 12, a region associated with various genetic disorders including Noonan syndrome and Trisomy 12p .

What types of BRAP antibodies are available for research?

Multiple BRAP antibodies have been developed for research purposes. These include:

• Mouse monoclonal IgG1 kappa light chain antibodies (such as D-5)

• Recombinant monoclonal antibodies generated against specific fragments (e.g., 100-250 amino acid fragment encoded by the human C6orf89 gene)

These antibodies are available in non-conjugated forms and various conjugated forms including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates .

What experimental applications are BRAP antibodies suitable for?

BRAP antibodies have been validated for multiple experimental applications including:

• Western blotting (WB)

• Immunoprecipitation (IP)

• Immunofluorescence (IF)

• Immunohistochemistry (IHC)

• Enzyme-linked immunosorbent assay (ELISA)

• Co-immunoprecipitation for protein interaction studies

Different antibodies may have species-specific applications. For example, some recombinant monoclonal antibodies have been reported to work for Western blotting with both human and mouse tissues but only for IHC with human samples .

How should BRAP antibodies be validated for specificity in tissue samples?

Validation of BRAP antibodies requires multi-approach confirmation:

• Knockout/knockdown controls: Compare staining between wild-type and BRAP knockout/knockdown samples. For example, studies have confirmed antibody specificity by showing no BRAP signals in lung tissues from BC004004−/− mice while detecting appropriate signals in wild-type mice .

• Western blot validation: Confirm the antibody detects bands of expected molecular weight (~65 kDa) that disappear in knockout models.

• Cross-reactivity testing: Test the antibody across multiple species to determine cross-reactivity limits. Some BRAP antibodies work with human, mouse, and rat samples, while others have species limitations .

• Multiple detection methods: Validate using both immunoblotting and immunostaining techniques to ensure consistent results across methodologies .

What are the optimal protocols for BRAP detection in immunofluorescence studies?

For optimal immunofluorescence detection of BRAP:

• Fixation: Use freshly prepared 4% paraformaldehyde in neutral PBS for 15 minutes at room temperature.

• Permeabilization: Incubate samples in 0.1% Triton X-100 in PBS for 15 minutes at room temperature.

• Blocking: Use 5% BSA for 30 minutes at 37°C.

• Primary antibody incubation: Dilute BRAP antibody 1:200 in blocking buffer and incubate in a humidified chamber overnight at 4°C.

• Washing: Perform three 5-minute washes with PBS-T.

• Secondary antibody incubation: Use appropriate fluorescent secondary antibodies (e.g., Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit) at 1:200 dilution for 1 hour at room temperature in the dark.

• Nuclear counterstaining: Use DAPI at 1:10 dilution.

• Mounting: Apply mounting media before visualization .

How can co-localization of BRAP with other proteins be effectively demonstrated?

For effective co-localization studies:

• Double immunofluorescence procedure:

  • Use primary antibodies from different host species (e.g., rabbit anti-BRAP and mouse anti-target protein).

  • Apply both primary antibodies simultaneously at appropriate dilutions (typically 1:200).

  • Use spectrally distinct secondary antibodies (e.g., Alexa Fluor 488 for BRAP and Alexa Fluor 594 for the target protein).

  • Look for orange/yellow signals in merged images indicating co-distribution.

• Controls required:

  • Single-stained controls to verify absence of spectral bleed-through.

  • Negative controls omitting primary antibodies.

  • Positive controls with known interaction partners.

For example, studies have successfully demonstrated co-localization of BRAP with S100A4 in the cytoplasm of fibroblasts using this approach .

How can BRAP antibodies be used to investigate protein-protein interactions?

BRAP antibodies can be used to study protein-protein interactions through several approaches:

• Co-immunoprecipitation (Co-IP):

  • Use BRAP antibodies conjugated to agarose or other solid supports.

  • Lyse cells under non-denaturing conditions.

  • Immunoprecipitate BRAP and associated proteins.

  • Analyze by Western blotting with antibodies against suspected interaction partners.

  This approach has successfully identified interactions between BRAP and proteins like ATG5 .

• Pull-down assays:

  • Express recombinant tagged BRAP (e.g., MBP-tagged C-terminal domain).

  • Express potential interaction partners with different tags (e.g., GST-tagged ATG5).

  • Perform pull-down experiments with appropriate affinity resins.

  • Detect interactions by Western blotting with BRAP antibodies.

  This approach confirmed direct binding between BRAP and ATG5 .

• Yeast two-hybrid screening:

  • Use BRAP as bait to identify potential interaction partners.

  • Verify interactions through secondary methods like Co-IP or pull-down assays.

  This approach identified over 300 potential BRAP-interacting proteins, including ATG5 and Rabin8 .

What roles does BRAP play in tissue-specific pathologies, and how can antibodies help investigate these functions?

BRAP is implicated in multiple tissue-specific pathologies that can be investigated using antibodies:

• Liver pathology:

  • BRAP deletion in liver causes disruption of normal architecture and inflammation.

  • BRAP regulates hepatocyte turnover via the Hippo pathway and YAP-mediated transcription.

  • Antibodies can be used to track BRAP expression in models of liver disease and correlate with fibrotic and inflammatory markers .

• Pulmonary fibrosis:

  • BRAP homologous protein deficiency attenuates bleomycin-induced pulmonary inflammation.

  • Antibodies can be used to monitor BRAP expression in fibrotic lung tissues and interstitial cells.

  • Immunofluorescence with BRAP antibodies can identify cell-specific expression patterns in fibrotic lesions .

• Airway cilia abnormalities:

  • BRAP knockout leads to abnormal tracheal cilia in mice.

  • BRAP interacts with Rabin8, affecting cilia development.

  • Antibodies can be used to co-localize BRAP with cilia markers like acetylated α-tubulin .

• Neuronal function:

  • BRAP signals have been detected in neurons of brain tissue samples.

  • Chronic unpredictable mild stress evokes more significant depressive-like effects in BRAP-deficient mice.

  • Antibodies can help track BRAP expression in specific neuronal populations .

How can BRAP antibodies be used to investigate autophagy mechanisms?

BRAP antibodies can be instrumental in investigating autophagy through several experimental approaches:

• Monitoring BRAP-ATG5 interactions:

  • ATG5 is a key autophagy protein that directly interacts with BRAP.

  • Use co-immunoprecipitation with BRAP antibodies to pull down ATG5 and assess how various cellular conditions affect this interaction.

  • Western blotting can quantify changes in BRAP-ATG5 complexes during autophagy induction or inhibition .

• Investigating autophagic activity in BRAP-deficient models:

  • Enhanced autophagic activity in fibroblasts due to lack of BRAP might contribute to resistance to pulmonary fibrosis.

  • Use BRAP antibodies alongside autophagy markers (LC3-II, p62) to correlate BRAP expression with autophagic flux.

  • Immunofluorescence can visualize co-localization between BRAP and autophagosomal structures .

• Examining the regulatory role of BRAP in autophagy:

  • BRAP functions as an E3 ubiquitin ligase that may regulate autophagy proteins through ubiquitination.

  • Use BRAP antibodies in ubiquitination assays to identify potential autophagy-related substrates.

  • Analyze how BRAP knockdown affects levels and post-translational modifications of autophagy components .

What are common issues with BRAP antibodies in Western blotting and how can they be resolved?

Common issues and solutions for Western blotting with BRAP antibodies include:

• Multiple bands or unexpected molecular weight:

  • BRAP typically appears as two bands of similar molecular weight at ~65 kDa.

  • Verify specificity using knockout/knockdown controls.

  • Optimize sample preparation to reduce protein degradation by adding protease inhibitors.

  • Adjust antibody concentration and incubation conditions.

• Weak or no signal:

  • Increase protein loading (25-50 μg total protein recommended).

  • Optimize transfer conditions for high molecular weight proteins.

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

  • Use more sensitive detection methods (e.g., enhanced chemiluminescence).

• High background:

  • Increase blocking time or concentration (5% BSA or non-fat milk).

  • Add 0.1-0.3% Tween-20 to washing buffer.

  • Optimize antibody dilution (typically 1:200 to 1:1000 for BRAP antibodies).

  • Ensure thorough washing between antibody incubations .

How can BRAP detection be optimized in different tissue types?

Optimization strategies for BRAP detection across different tissues include:

• Liver tissues:

  • Perfusion fixation recommended for better preservation of liver architecture.

  • Mild antigen retrieval methods (citrate buffer, pH 6.0) to preserve protein epitopes.

  • Higher antibody concentrations may be needed due to high protein content .

• Lung tissues:

  • Inflation fixation techniques improve preservation of airway structures.

  • Careful permeabilization to maintain delicate alveolar structures.

  • Background can be reduced with extended blocking (2-3 hours) .

• Brain tissues:

  • Transcardial perfusion fixation essential for good morphology.

  • Extended antibody incubation times (48-72 hours at 4°C) for better penetration.

  • Use of detergents like Triton X-100 (0.1-0.3%) to improve antibody access .

• Cell culture:

  • Milder fixation protocols (2-4% paraformaldehyde for 10-15 minutes).

  • Brief permeabilization (0.1% Triton X-100 for 5-10 minutes).

  • Shorter antibody incubation times can be effective (1-2 hours at room temperature) .

What are key considerations when using BRAP antibodies in disease models?

When using BRAP antibodies in disease models, researchers should consider:

• Altered protein expression levels:

  • Disease states may significantly up or downregulate BRAP expression.

  • Adjust antibody concentrations accordingly to avoid saturation or insufficient signal.

  • Include appropriate disease controls alongside normal tissue controls.

• Modified protein characteristics:

  • Post-translational modifications may affect antibody binding.

  • Consider using multiple antibodies recognizing different epitopes.

  • Verify findings with complementary techniques like qRT-PCR or mass spectrometry.

• Background interference:

  • Inflammatory or fibrotic tissues often show higher non-specific binding.

  • Include isotype controls to distinguish specific from non-specific signals.

  • Use tissue clearing techniques for thick sections with high autofluorescence .

How can BRAP antibodies contribute to understanding cellular signaling pathways?

BRAP antibodies can provide valuable insights into cellular signaling through:

• MAP kinase pathway regulation:

  • BRAP negatively regulates MAP kinase activity through Ksr-1 scaffold protein inactivation.

  • Use BRAP antibodies to track changes in BRAP-Ksr1 interactions under various stimuli.

  • Correlate BRAP localization with activation status of downstream MAP kinase components.

• Hippo pathway modulation:

  • BRAP deletion affects YAP-mediated transcription.

  • Antibodies can help analyze nuclear translocation of YAP in relation to BRAP expression.

  • Co-immunoprecipitation can identify components of BRAP-Hippo pathway complexes .

• Ras signaling interactions:

  • BRAP functions as a Ras-responsive E3 ubiquitin ligase.

  • Antibodies can be used to study how Ras activation affects BRAP ubiquitination activity.

  • Proximity ligation assays can visualize BRAP-Ras interactions in situ .

What are potential applications of BRAP antibodies in developing targeted therapeutics?

BRAP antibodies can support therapeutic development through:

• Validation of BRAP as a drug target:

  • BRAP's role in liver morphology, pulmonary fibrosis, and cilia function suggests it could be a therapeutic target.

  • Antibodies can help validate target engagement of small molecule BRAP inhibitors.

  • Immunohistochemistry can assess BRAP expression patterns in patient samples to identify responsive populations .

• Development of antibody-drug conjugates (ADCs):

  • If BRAP has cell-surface exposure in certain disease states, it could potentially be targeted with ADCs.

  • Research-grade antibodies can serve as starting points for therapeutic antibody development.

  • Streamlined analytical workflows can characterize antibody binding and specificity .

• Monitoring treatment response:

  • Antibodies can assess changes in BRAP expression or localization during treatment.

  • Quantitative image analysis of BRAP immunostaining could serve as a pharmacodynamic biomarker.

  • Sequential tissue sampling could track treatment-induced changes in BRAP pathway activation .

How might rational antibody design approaches be applied to develop novel BRAP-targeting antibodies?

Advanced antibody design strategies could enhance BRAP research through:

• Epitope-specific antibody design:

  • Design complementary peptides targeting specific epitopes within BRAP.

  • Graft these peptides onto antibody CDR regions to create highly specific antibodies.

  • This rational approach could generate antibodies binding to functional domains of BRAP with greater specificity than conventional methods .

• Multi-loop design strategies:

  • Engineer antibodies with multiple complementary peptides in different CDR loops.

  • This could increase binding affinity and specificity for BRAP.

  • The cooperative binding of multiple loops could enable targeting of specific BRAP conformations or interaction interfaces .

• Application to disordered regions:

  • If BRAP contains intrinsically disordered regions, rational design methods could generate antibodies specifically targeting these challenging epitopes.

  • Such antibodies could provide insights into the structural dynamics of BRAP and its interactions .

What are emerging approaches for studying BRAP in complex tissue environments?

Advanced techniques for BRAP analysis in complex tissues include:

• Spatial transcriptomics integration:

  • Combine BRAP immunostaining with spatial transcriptomics to correlate protein localization with gene expression patterns.

  • This could reveal regulatory relationships between BRAP and other genes in specific tissue microenvironments.

• Multiplex immunofluorescence:

  • Simultaneously visualize BRAP with multiple markers of cellular identity and function.

  • This approach could map BRAP expression across diverse cell types and states within tissues.

  • Particularly valuable for understanding BRAP's role in heterogeneous environments like liver and lung .

• 3D tissue imaging:

  • Apply clearing techniques and volume imaging to visualize BRAP distribution in intact tissue volumes.

  • This could reveal spatial relationships between BRAP-expressing cells and anatomical features like blood vessels or airways .

How might single-cell approaches enhance understanding of BRAP function?

Single-cell technologies can advance BRAP research through:

• Single-cell protein analysis:

  • Apply mass cytometry or single-cell Western blotting to quantify BRAP across individual cells.

  • Correlate BRAP levels with cell state markers to identify functional relationships.

  • Characterize heterogeneity in BRAP expression within seemingly homogeneous populations.

• Spatial single-cell analysis:

  • Combine BRAP immunostaining with single-cell RNA sequencing to correlate protein expression with transcriptional profiles.

  • This could identify gene networks associated with high or low BRAP expression.

  • Particularly valuable for understanding BRAP's role in specific cell subtypes within complex tissues .

• Live-cell imaging of BRAP dynamics:

  • Develop fluorescent protein fusions or nanobodies to track BRAP localization and interactions in living cells.

  • This could reveal dynamic responses to cellular stresses or signaling events.

  • May provide insights into BRAP's role in processes like autophagy or cilia formation .