SMARCC2 Antibody, HRP conjugated

What is SMARCC2 and why is it an important research target?

SMARCC2 is a core subunit of the SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complex. It plays a key role in the early assembly of this complex and contributes to its fundamental chromatin remodeling function. SMARCC2 has gained research interest because it functions as a tumor suppressor in multiple cancers, particularly in glioblastoma and potentially in ovarian cancer . Its expression is notably lower in high-grade gliomas compared to low-grade gliomas, suggesting its relevance as a prognostic marker . SMARCC2's ability to inhibit cancer cell proliferation, migration, and invasion makes it a valuable target for understanding cancer pathogenesis and developing potential therapeutic approaches.

What are the primary signaling pathways affected by SMARCC2?

SMARCC2 has been shown to inhibit the Wnt/β-catenin signaling pathway in glioma cells. Research demonstrates that SMARCC2 downregulates the expression of N-cadherin, vimentin, Snail, and β-catenin while upregulating T-cadherin expression . Additionally, SMARCC2 has been found to negatively regulate DKK1 transcription by dynamically regulating chromatin structure and closing the promoter region of DKK1, which can be bound by the transcription factor EGR1. Through this mechanism, SMARCC2 can inhibit the PI3K-AKT pathway, as DKK1 knockdown was shown to reduce glioblastoma cell proliferation through this signaling axis .

What dilutions are recommended for SMARCC2 antibodies in different applications?

Based on available product information, the following dilutions are generally recommended for SMARCC2 antibodies, though specific optimization may be required for HRP-conjugated versions:

These recommendations provide starting points for assay optimization. The actual working concentration may vary and should be determined experimentally by each researcher based on their specific samples and conditions .

How should researchers validate SMARCC2 antibody specificity?

Proper validation of SMARCC2 antibodies should include multiple approaches:

• Positive and negative control samples: Use cell lines or tissues with known high expression (low-grade gliomas) and low expression (high-grade gliomas) of SMARCC2 .

• Knockdown/overexpression validation: Compare antibody signal in samples with SMARCC2 knockdown using siRNA (e.g., siSMARCC2-1, siSMARCC2-3) and overexpression using adenovirus vectors carrying SMARCC2 cDNA .

• Molecular weight verification: Confirm detection at the expected molecular weight. Note that while the calculated molecular weight of SMARCC2 is approximately 133 kDa, the observed molecular weight in some experiments is approximately 72 kDa . This discrepancy should be considered during validation.

• Cross-reactivity assessment: Verify specificity across species if working with both human and mouse samples, as some antibodies are reactive to both species .

How can researchers effectively design co-immunoprecipitation experiments to study SMARCC2 protein interactions?

When designing co-immunoprecipitation (Co-IP) experiments to study SMARCC2 interactions, researchers should consider the following methodological approach:

• Cell preparation: Seed approximately 2×10^5 cells/dish and incubate for 24 hours to ensure adequate expression levels .

• Protein extraction and quantification: Extract proteins under non-denaturing conditions to preserve native protein-protein interactions.

• Co-IP procedure: Use equal amounts of protein for immunoprecipitation with rabbit anti-SMARCC2 antibody (1:1000 dilution) at 4°C overnight, followed by incubation with protein A/G sepharose magnetic beads (40 μl) at 4°C .

• Washing and elution: Wash with appropriate lysis buffer and separate the protein complex by magnetic separation. Resuspend in SDS buffer for subsequent analysis .

• Detection of interacting partners: Perform western blotting to detect known or suspected interacting partners such as c-Myc, which has been shown to associate with SMARCC2. Consider probing for components of the Wnt/β-catenin pathway or DKK1-related factors based on SMARCC2's known regulatory roles .

What experimental approach should be used to study SMARCC2's impact on cell migration and invasion?

To investigate SMARCC2's effects on cell migration and invasion, researchers can employ the following methodology:

• Cell preparation: For migration assays, establish SMARCC2 knockdown using siRNAs (siSMARCC2-1/3) or overexpression using adenovirus vectors carrying SMARCC2 cDNA .

• Wound healing assay: Create a scratch in a confluent cell monolayer and measure wound closure over time to assess migration. Compare results between control, knockdown, and overexpression groups .

• Transwell invasion assay: Seed 1×10^5 glioma cells (such as U87MG, T98G, or LN229) in serum-free medium in the upper chamber of Transwell plates precoated with Matrigel. Place medium with 10% FBS in the lower chamber as a chemoattractant .

• Analysis: After 6-8 hours of incubation, remove non-invasive cells, fix migratory/invasive cells with 20% methanol for 5 minutes, and stain with 0.1% crystal violet for 5 minutes. Observe cells from six randomly selected fields under a confocal microscope at 200× magnification and quantify using ImageJ software .

• Marker analysis: Perform parallel western blotting to assess changes in EMT markers (N-cadherin, vimentin, Snail, β-catenin, T-cadherin) to correlate with migration/invasion phenotypes .

How can researchers investigate the role of SMARCC2 domains in chromatin remodeling?

Studies have shown that the SWIRM and SANT domains of SMARCC2 have differential contributions to its chromatin remodeling function, with the SWIRM domain playing a more critical role . To investigate domain-specific functions, researchers should:

• Generate domain-specific constructs: Create expression vectors containing full-length SMARCC2 or constructs with deletions or mutations in specific domains (SWIRM, SANT).

• Functional assays: Perform chromatin accessibility assays (e.g., ATAC-seq) to determine how different domain mutants affect chromatin states at target genes like DKK1.

• Promoter binding analysis: Use chromatin immunoprecipitation (ChIP) assays to assess how domain mutations affect SMARCC2 binding to target gene promoters, particularly those bound by transcription factors like EGR1 .

• Transcriptional output: Measure expression levels of target genes using RT-qPCR following expression of domain mutants to correlate chromatin changes with transcriptional outcomes.

What are common issues when using HRP-conjugated antibodies and how can they be addressed?

When working with HRP-conjugated SMARCC2 antibodies, researchers may encounter several technical challenges:

• High background:

  • Cause: Insufficient blocking, high antibody concentration, or sample overloading

  • Solution: Optimize blocking conditions (use 5% BSA or milk), reduce antibody concentration, and ensure appropriate protein loading

• Weak or no signal:

  • Cause: Protein degradation, insufficient antigen, epitope masking, or antibody degradation

  • Solution: Use fresh samples, optimize protein extraction protocol, consider different fixation methods for ICC/IF, and store antibody properly (at -20°C long-term, 4°C for up to one month)

• Non-specific bands:

  • Cause: Cross-reactivity with similar epitopes or protein degradation

  • Solution: Include appropriate controls (SMARCC2 knockdown/overexpression samples), optimize antibody dilution, and use freshly prepared samples

• Signal variability:

  • Cause: Inconsistent technique or reagent instability

  • Solution: Standardize protocols, avoid repeated freeze-thaw cycles of antibody , and prepare fresh working solutions for each experiment

What controls should be included in SMARCC2 antibody experiments?

For rigorous SMARCC2 antibody experiments, include the following controls:

• Positive control: Samples known to express SMARCC2 (e.g., low-grade glioma tissue or cell lines with confirmed SMARCC2 expression)

• Negative control:

  • Technical negative: Primary antibody omission

  • Biological negative: Samples with SMARCC2 knockdown using validated siRNAs (e.g., siSMARCC2-1, siSMARCC2-3)

• Overexpression control: Cells transfected with SMARCC2 expression vectors

• Loading control: For western blots, include housekeeping proteins like GAPDH (1:3,000) or β-actin (1:3,000)

• Isotype control: Include rabbit IgG control at the same concentration as the SMARCC2 antibody to assess non-specific binding

How does SMARCC2 expression correlate with clinical outcomes in different cancers?

SMARCC2 expression has significant implications for cancer prognosis and clinical outcomes:

• Glioma: SMARCC2 mRNA and protein expression levels are significantly higher in low-grade glioma tissues compared to high-grade glioma tissues. Bioinformatics analysis has revealed that upregulated expression of SMARCC2 is associated with more favorable prognosis in patients with glioma .

• Glioblastoma: As a tumor suppressor in glioblastoma, reduced SMARCC2 expression correlates with more aggressive disease. Overexpression of SMARCC2 in experimental models significantly inhibited the size of intracranial gliomas in nude mice, suggesting its potential therapeutic value .

• Ovarian cancer: Gene amplification of SMARCC2 has been reported in ovarian high-grade serous carcinoma (OHGSC), though the complete clinical implications are still being investigated .

Researchers should consider these expression patterns when selecting appropriate model systems and interpreting experimental results.

What is the relationship between SMARCC2 and epithelial-mesenchymal transition in cancer?

SMARCC2 plays a significant role in regulating epithelial-mesenchymal transition (EMT), a process critical for cancer progression and metastasis:

• Marker regulation: SMARCC2 overexpression significantly downregulates the expression of mesenchymal markers (N-cadherin, vimentin, Snail, β-catenin) while upregulating epithelial markers (T-cadherin) .

• Functional consequences: By modulating EMT status, SMARCC2 inhibits the migration and invasion capabilities of glioblastoma cell lines .

• Mechanistic pathway: This regulation occurs through SMARCC2's interaction with c-Myc, leading to downregulation of c-Myc expression and subsequent inhibition of the Wnt/β-catenin signaling pathway, which is a key driver of EMT .

• Experimental verification: Western blotting and immunofluorescence analyses have confirmed these changes in EMT marker expression following SMARCC2 manipulation, providing a methodological framework for researchers investigating similar questions .