BRCC3 Antibody

What is BRCC3 and what are its key cellular functions?

BRCC3, also known as BRCC36, is a subunit of the BRCA1-BRCA2-containing complex (BRCC), functioning as an E3 ubiquitin ligase. This 36 kDa protein plays essential roles in several critical cellular processes:

• DNA damage response, where it contributes to the stable accumulation of BRCA1 at DNA break sites

• Cell cycle control, particularly in G2/M phase transition in breast cancer cells

• Deubiquitination activity, specifically cleaving Lys-63-linked polyubiquitin chains

• Regulation of inflammatory responses through deubiquitination of NLRP3, promoting inflammasome activation

• Maintenance of genomic stability, preventing accumulation of genetic mutations that can lead to cancer development

The importance of BRCC3 in these pathways makes it a promising therapeutic target for enhancing chemotherapy and radiation therapy efficacy in cancer patients .

What applications are BRCC3 antibodies suitable for?

BRCC3 antibodies have been validated for multiple research applications, with varying optimal dilutions for different techniques:

It is highly recommended to optimize antibody concentrations for each specific experimental system and sample type to achieve optimal results .

How should researchers validate the specificity of BRCC3 antibodies?

Validating antibody specificity is crucial for obtaining reliable research data. For BRCC3 antibodies, implement these comprehensive validation approaches:

• Positive and negative controls: Include lysates from cells known to express BRCC3 (such as HeLa or PC-3) as positive controls, and implement negative controls through:

  • BRCC3 knockout cell lines generated via CRISPR-Cas9 (as described in T24 bladder cancer cells)

  • siRNA or shRNA-mediated BRCC3 knockdown samples

  • Pre-absorption with the immunizing peptide (when available)

• Cross-reactivity testing: Confirm reactivity with target species (human, mouse, rat) and evaluate potential cross-reactivity with related proteins .

• Multiple detection methods: Verify results using at least two independent techniques (e.g., WB and IHC or IF).

• Molecular weight verification: Confirm that the detected protein appears at the expected molecular weight (~35-36 kDa for BRCC3).

• Peptide competition assay: Pre-incubate the antibody with the immunogen peptide prior to application to confirm signal specificity .

What are the optimal storage and handling conditions for BRCC3 antibodies?

Proper storage and handling of BRCC3 antibodies are essential for maintaining their activity and specificity:

What methodologies are most effective for studying BRCC3's role in DNA repair pathways?

To investigate BRCC3's functions in DNA repair mechanisms, researchers should consider these advanced methodological approaches:

• DNA damage induction and repair kinetics analysis:

  • Treat cells with DNA-damaging agents (ionizing radiation, etoposide, or cisplatin)

  • Monitor BRCC3 recruitment to damage sites using immunofluorescence with time-course analysis

  • Quantify γ-H2AX foci formation and resolution in BRCC3-depleted versus control cells

  • Measure DNA repair efficiency using comet assay or NHEJ/HR reporter assays

• Protein complex analysis:

  • Use proximity ligation assays (PLA) to visualize BRCC3 interactions with BRCA1/BRCA2 complexes in situ

  • Perform sequential ChIP experiments to identify BRCC3 co-localization with repair factors at damaged chromatin

  • Apply APEX2-based proximity labeling to identify novel BRCC3 interaction partners at damage sites

• Functional deubiquitination studies:

  • Conduct in vitro deubiquitination assays using purified BRCC3 and K63-linked ubiquitin chains

  • Monitor changes in ubiquitination status of known substrates in BRCC3-depleted cells using ubiquitin-specific antibodies

  • Engineer catalytically inactive BRCC3 mutants to distinguish enzymatic from scaffolding functions

How can researchers generate and validate BRCC3 knockout models?

Generating reliable BRCC3 knockout models requires careful experimental design:

• CRISPR-Cas9 mediated knockout strategy:

  • Design multiple sgRNAs targeting early exons of BRCC3 (example from research: BRCC3-specific sgRNAs cloned into lenti-v2)

  • Package lentiviral vectors in HEK293T cells with appropriate packaging vectors

  • Transduce target cells (e.g., T24 bladder cancer cells) with lentivirus

  • Select transduced cells with appropriate antibiotic (e.g., 600 ng/ml puromycin for 5 days)

  • Isolate single clones using limiting dilution method in 96-well plates

  • Allow clonal expansion for approximately 14 days

• Validation of knockout efficiency:

  • Confirm deletion at genomic level using PCR and sequencing

  • Verify protein loss by immunoblotting with validated anti-BRCC3 antibodies

  • Perform functional validation by assessing known BRCC3-dependent pathways

  • Examine phenotypic characteristics (growth rate, cell cycle distribution, response to DNA damage)

• Controls and complementation:

  • Include non-targeting sgRNA controls

  • Generate rescue cell lines by re-expressing BRCC3 to confirm phenotype specificity

  • Consider generating domain-specific mutants to dissect functions

What techniques can best elucidate BRCC3's role in cancer progression and therapy resistance?

Investigating BRCC3's contributions to cancer pathogenesis and treatment response requires multi-faceted approaches:

• Clinical correlation studies:

  • Analyze BRCC3 expression in tumor vs. normal tissues using IHC with optimized protocols (TE buffer pH 9.0 or citrate buffer pH 6.0 for antigen retrieval)

  • Correlate expression levels with patient survival, tumor grade, and treatment response metrics

  • Perform multivariate analysis to determine if BRCC3 is an independent prognostic factor

• Therapy resistance mechanisms:

  • Generate therapy-resistant cell lines and analyze BRCC3 expression/activity changes

  • Conduct combination studies with BRCC3 depletion plus standard chemotherapy or radiation

  • Measure DNA damage accumulation and repair kinetics in resistant vs. sensitive cells with varying BRCC3 levels

• In vivo tumor models:

  • Establish xenograft models with BRCC3-modulated cancer cells

  • Monitor tumor growth, metastatic potential, and therapy response

  • Analyze tumor microenvironment changes related to BRCC3 expression

• Signaling pathway analysis:

  • Investigate BRCC3-dependent NF-κB activation through TRAF2 binding using co-immunoprecipitation

  • Assess nuclear translocation of NF-κB subunits in BRCC3-depleted cells

  • Measure expression of NF-κB target genes using qRT-PCR or RNA-seq

  • Evaluate effects of NF-κB inhibitors in BRCC3-overexpressing cancer models

How can researchers investigate BRCC3's role in inflammasome activation?

To explore BRCC3's function in regulating inflammasome activity, particularly through NLRP3 deubiquitination:

• NLRP3 inflammasome activation assays:

  • Prime cells with LPS followed by NLRP3 activators (ATP, nigericin, or crystals)

  • Compare inflammasome assembly in BRCC3-sufficient and BRCC3-deficient cells

  • Measure IL-1β and IL-18 secretion by ELISA

  • Detect caspase-1 activation and pyroptosis markers

• Deubiquitination analysis:

  • Immunoprecipitate NLRP3 and assess its ubiquitination status in the presence/absence of BRCC3

  • Perform in vitro deubiquitination assays with purified components

  • Use ubiquitin chain-specific antibodies to distinguish between different ubiquitin linkages

• Interaction studies:

  • Map the interaction domains between BRCC3 and NLRP3 using truncation mutants

  • Visualize co-localization using confocal microscopy with dual immunofluorescence

  • Apply FRET or BiFC techniques to confirm direct interaction in living cells

• Stimulus-specific regulation:

  • Compare the requirement for BRCC3 across different NLRP3 activation pathways

  • Assess whether BRCC3 participates in other inflammasome complexes (AIM2, NLRC4)

  • Investigate upstream regulators of BRCC3 recruitment to inflammasomes

What experimental approaches are optimal for studying BRCC3's cell cycle regulatory functions?

To investigate BRCC3's impact on cell cycle progression, particularly its association with G2/M arrest:

• Cell cycle synchronization and analysis:

  • Synchronize cells using methods appropriate for specific cell cycle phases (double thymidine block, nocodazole, etc.)

  • Perform flow cytometry with propidium iodide or DAPI staining to quantify cell cycle distribution

  • Use BrdU incorporation assays to measure S-phase progression

  • Apply phospho-histone H3 staining to specifically identify mitotic cells

• Mitotic checkpoint regulation:

  • Assess BRCC3 interactions with key cell cycle regulators through co-immunoprecipitation

  • Monitor checkpoint activation after DNA damage in BRCC3-depleted cells

  • Examine localization of BRCC3 during different cell cycle phases using immunofluorescence

  • Analyze spindle assembly and chromosome alignment in BRCC3-deficient cells

• Cyclin-dependent kinase (CDK) activity:

  • Measure CDK1/Cyclin B activity in BRCC3-modulated cells

  • Assess phosphorylation status of CDK substrates

  • Determine if BRCC3 is itself regulated by cell cycle-dependent phosphorylation

• Live cell imaging:

  • Use fluorescent cell cycle reporters in BRCC3-depleted cells

  • Track mitotic duration and abnormalities through time-lapse microscopy

  • Quantify cell division defects and mitotic catastrophe events