BRN2 Antibody

Validating BRN2 Antibody Specificity in Neural Crest-Derived Cancers

Experimental Design:

• Perform parallel Western blotting using cell lysates from BRN2-knockout (CRISPR/Cas9) and wild-type melanoma/prostate cancer lines . Validate with siRNA knockdown (≥70% efficiency) followed by qRT-PCR to confirm BRN2 mRNA reduction .

• Use tissues from Brn2 conditional knockout mice (e.g., Tyr::CreER;Brn2fl/fl) as negative controls in IHC .

Critical Parameters:

Optimizing BRN2 Immunoprecipitation for Chromatin Studies

Methodological Approach:

• Pre-clearing: Use protein A/G magnetic beads with 1% BSA in RIPA buffer to reduce nonspecific binding .

• Crosslinking: Combine 1% formaldehyde (20 min) with 2.5 mM DSG (30 min) to preserve protein-DNA interactions .

• Elution: Competitor peptides matching the BRN2 epitope (e.g., residues 393-C terminus) improve yield .

Common Pitfalls:

• Overfixation (>30 min formaldehyde) masks epitopes in the POU domain .

• Protease inhibitors must include 10 mM NEM to prevent deubiquitination .

Resolving Discrepant BRN2 Localization Across Studies

Conflict Analysis:

• Nuclear vs. Cytoplasmic Staining: Check fixation method; methanol fixation preserves nuclear localization better than PFA in melanoma cells .

• Tissue-Specific Expression: Use RNAscope® to correlate protein (IHC) and mRNA (ISH) levels, especially in neural/neuroendocrine tumors .

Case Example:
In prostate cancer, nuclear BRN2 correlates with NE markers (SYP: r = 0.271, p < 0.0001), while cytoplasmic staining associates with EMT (VIM upregulation) .

Designing BRN2 Studies in Therapy-Resistant Metastases

Integrated Workflow:

• Model Selection: Use PDX models from enzalutamide-resistant CRPC (e.g., 42F ENZR) or BRAFi-resistant melanoma .

• Temporal Sampling: Collect samples at baseline, 48h, and 7d post-treatment to capture BRN2 dynamics .

• Multiplex Assays: Combine ChIP-seq (H3K27ac), CUT&RUN (BRN2), and scRNA-seq to map regulatory networks.

Key Findings Table:

Reconciling BRN2’s Dual Roles in Apoptosis and DNA Repair

Contradiction Resolution Framework:

• Context Dependency: In UV-treated melanoma, BRN2 binds PARP1 (Kd = 12.3 nM) to enhance NHEJ repair .

• Transcriptional vs. Non-Canonical Roles:

  • CRISPRa (dCas9-VPR) at ASCL1 locus → BRN2-dependent neurogenesis

  • BRN2-Ku80 interaction → error-prone repair (γH2AX foci ↑ 3.1-fold)

Experimental Validation:

• Co-IP with anti-BRN2 (B-2 clone) and anti-PARP1 (46D11), followed by proximity ligation assay .

Multimodal BRN2 Detection in Single-Cell Analyses

Innovative Workflow:

• Spatial Transcriptomics: CODEX® multiplexing with BRN2 (Cy5), AR (AF488), and SYP (AF594) .

• CITE-seq: BRN2 antibody-oligo conjugates (TotalSeq™-B) paired with 10x Genomics .

Optimization Notes:

• Heat-induced epitope recovery (HIER) at 95°C for 20 min in pH 8.0 EDTA improves CITE-seq signal .

• Antibody concentration: 0.25 µg/ml for CODEX®, 1.0 µg/ml for CITE-seq .

Targeting BRN2 in Neuroendocrine Plasticity

Pharmacological Synergy:

• BRN2 inhibitor B18-94 (IC50 = 2.04 µM) + enzalutamide reduces tumor volume by 68% vs. monotherapy in 42F PDX .

• Resistance mechanism: BRN2 T306A mutation disrupts drug binding (ΔΔG = +4.7 kcal/mol) .

Combination Screening:

Correlating BRN2 Isoforms with Clinical Outcomes

Isoform-Specific PCR:

• Primer Design:

  • Isoform 1 (NM_005604): F-5′-CGGACCTCAACAGCCTCTAC-3′, R-5′-GCCTCGGTAGTGGTCTTGAT-3′

  • Isoform 2 (NM_001369356): F-5′-GCTGGAGCCTTCAGAAGGAC-3′, R-5′-CAGGGTCCTGGTAGGTGTCT-3′

TCGA Analysis:

• Isoform 2 dominance correlates with worse OS in melanoma (HR = 2.31, p = 0.008) .

Methodological Resources

BRN2 Antibody Validation Table:

Statistical Considerations:

• For IHC quantification, use H-score: (3×% strong) + (2×% moderate) + (1×% weak). Significant if ≥160 .

• ChIP-seq peak calling: MACS2 with q < 0.01 and fold-enrichment >4 vs. IgG .