SMARCC1 Monoclonal Antibody

What is SMARCC1 and what cellular functions does it regulate?

SMARCC1/BAF155 is a key component of the SWI/SNF chromatin remodeling complex essential for regulating gene expression by remodeling chromatin structure and influencing transcriptional activities. It belongs to both the neural progenitors-specific chromatin remodeling complex (npBAF complex) and the neuron-specific chromatin remodeling complex (nBAF complex) . As part of these complexes, SMARCC1 plays vital roles in modulating cellular processes including cell cycle progression, differentiation, and DNA damage response. The protein is crucial for normal cell function, and its dysregulation has been linked to various diseases, including cancer, developmental disorders, and neurological conditions . Understanding SMARCC1's function is particularly important as it demonstrates a dual regulatory role, functioning as both an oncogene and tumor suppressor in different cancer contexts .

What applications are SMARCC1 monoclonal antibodies typically used for in research?

SMARCC1 monoclonal antibodies are versatile research tools utilized across multiple laboratory techniques. The most common applications include:

• Western Blot (WB): Used at dilutions ranging from 1:500 to 1:16000 depending on the specific antibody and sample type .

• Immunohistochemistry (IHC): Typically used at dilutions between 1:50 and 1:1000 to visualize SMARCC1 expression in tissue sections .

• Immunoprecipitation (IP): Effective for isolating SMARCC1 and its interacting partners from cellular lysates .

• Chromatin Immunoprecipitation (ChIP): Used to study SMARCC1's interactions with chromatin and DNA, typically requiring 5μg antibody for 10-15μg of chromatin .

• Immunofluorescence/Immunocytochemistry (IF/ICC): Employed at dilutions of 1:50 to 1:200 to visualize subcellular localization .

• ELISA: For quantitative detection of SMARCC1 protein levels .

Each application requires specific sample preparation and antibody dilution optimization to achieve reliable and reproducible results.

What is the typical reactivity profile of SMARCC1 monoclonal antibodies?

Most commercially available SMARCC1 monoclonal antibodies demonstrate reactivity with human samples, with many also cross-reacting with mouse and rat specimens . For instance, the SMARCC1/BAF155 Rabbit Monoclonal Antibody (CAB4275) exhibits high reactivity and specificity with human samples and also shows reactivity with mouse samples . The antibody with catalog number 17722-1-AP has been validated to show reactivity with human, mouse, and rat samples in various applications . Within human tissues, positive immunohistochemistry detection has been reported in multiple tissue types including brain, kidney, ovary, placenta, testis, malignant melanoma, and appendicitis tissues . When planning experiments, it's crucial to verify the specific reactivity profile of your selected antibody clone to ensure compatibility with your experimental system.

How does SMARCC1 expression correlate with prognosis in different cancer types?

SMARCC1 demonstrates context-dependent roles in cancer progression and prognosis, with significant variations between cancer types:

Conversely, in bladder cancer (BC), high SMARCC1 expression shows a negative correlation with patient outcomes. IHC analysis of 54 BC tissue samples revealed that elevated SMARCC1 expression positively correlates with higher T stage. Kaplan-Meier survival analysis demonstrated that patients with high SMARCC1 expression had decreased survival rates compared to those with negative SMARCC1 expression .

In prostate cancer (PCa), SMARCC1 demonstrates tumor-suppressive properties. Knockdown of SMARCC1 in PCa cells significantly increases cell viability, proliferation, and migration while promoting epithelial-mesenchymal transition (EMT) . These findings suggest that SMARCC1 expression patterns and their prognostic implications must be carefully evaluated within the specific cancer context.

What experimental approaches can be used to study SMARCC1 function in cancer progression?

Investigating SMARCC1's role in cancer requires integrating multiple experimental approaches:

• RNA Interference and Gene Editing:

  • siRNA transfection (e.g., using sequences like 5′-CCUCACAAGACGAUGAAGATT-3′) for transient knockdown of SMARCC1 .

  • Lentiviral shRNA vectors for stable SMARCC1 knockdown, selected with puromycin (typically 2 μg/ml) .

  • CRISPR-Cas9 genome editing for complete knockout models.

• Functional Assays:

  • Colony formation assays to assess clonogenic potential .

  • Cell viability assays (e.g., CCK-8) to monitor proliferation after SMARCC1 manipulation .

  • Flow cytometry for cell cycle analysis and apoptosis assessment .

  • EdU incorporation assays to quantify cells in S phase .

  • Transwell and wound healing assays to evaluate migration capacity .

• Molecular Analysis:

  • qRT-PCR and western blotting to confirm knockdown efficiency and measure expression of downstream targets .

  • Analysis of EMT markers (E-cadherin, N-cadherin, vimentin) and EMT-related transcription factors (Slug, Snail, Zeb1) .

  • Evaluation of signaling pathway components (e.g., PI3K/AKT pathway) .

• In Vivo Models:

  • Subcutaneous xenograft models to assess tumor growth, with measurements calculated as V = (L × W²)/2 .

  • Lung metastasis models to evaluate metastatic potential .

  • Immunohistochemistry of tumor sections for SMARCC1, proliferation markers (Ki67), and prostate-specific markers (P504S) .

These methodologies collectively provide a comprehensive understanding of SMARCC1's functional impact on cancer cell behavior both in vitro and in vivo.

What mechanisms regulate SMARCC1 nuclear translocation and how does this impact its function?

SMARCC1's nuclear translocation is tightly regulated by nuclear transport proteins, which directly impacts its chromatin remodeling functions. Research has identified key regulators of this process:

KPNA2 (Karyopherin alpha 2) serves as a critical nuclear import receptor for SMARCC1. Alongside nucleoporins Nup50 and Nup153, KPNA2 regulates the process of SMARCC1 nuclear translocation in bladder cancer cells . This transport machinery recognizes nuclear localization signals on SMARCC1 and facilitates its movement through the nuclear pore complex.

The nuclear localization of SMARCC1 is essential for its function as part of the SWI/SNF chromatin remodeling complex. Only when properly localized to the nucleus can SMARCC1 participate in chromatin structure regulation, gene transcription control, cell cycle progression modulation, and DNA damage response . Disruption of this nuclear transport mechanism can lead to mislocalization of SMARCC1, potentially contributing to its altered function in cancer cells.

Understanding this regulatory process offers potential therapeutic opportunities. Targeting the KPNA2-mediated nuclear import of SMARCC1 could provide a novel approach for modulating SMARCC1 activity in cancers where its function is dysregulated. Research suggests that SMARCC1 may be a competent candidate as a diagnostic biomarker and therapeutic target in certain cancers based on these regulatory mechanisms .

What are the optimal conditions for Western blot detection of SMARCC1?

When performing Western blot analysis for SMARCC1 detection, researchers should follow these methodological guidelines for optimal results:

• Sample Preparation:

  • Lyse cells or tissues in RIPA buffer supplemented with protease inhibitors.

  • Use fresh samples when possible; if frozen, ensure rapid thawing to prevent protein degradation.

  • Load 20-50 μg of total protein per lane depending on SMARCC1 expression levels in your sample.

• Electrophoresis and Transfer:

  • SMARCC1 is a large protein (~155 kDa), requiring lower percentage (7-10%) SDS-PAGE gels for proper resolution.

  • Use wet transfer systems with cooling for optimal transfer of large proteins, running at 100V for 90-120 minutes.

• Antibody Selection and Dilution:

  • For the SMARCC1/BAF155 Rabbit Monoclonal Antibody (CAB4275), use at 1:500 to 1:2000 dilution .

  • For the 17722-1-AP antibody, recommended dilutions range from 1:2000 to 1:16000 .

  • Verify antibody specificity using positive controls such as K-562 cells, Jurkat cells, rat brain tissue, or rat testis tissue, which have been validated to express detectable levels of SMARCC1 .

• Incubation Parameters:

  • Primary antibody incubation should be performed overnight at 4°C for optimal signal-to-noise ratio.

  • Secondary antibody (anti-rabbit HRP-conjugated) can be incubated for 1 hour at room temperature.

  • Include adequate washing steps (3-5 times for 5-10 minutes each) between antibody incubations.

• Detection and Analysis:

  • Enhanced chemiluminescence (ECL) systems are suitable for SMARCC1 detection.

  • Exposure times should be optimized based on expression levels and antibody dilution.

  • Quantification should be normalized to appropriate loading controls (β-actin, GAPDH, or total protein).

These detailed parameters ensure consistent and reliable detection of SMARCC1 in Western blot applications across various sample types.

How should immunohistochemistry for SMARCC1 be optimized in different tissue types?

Successful immunohistochemical detection of SMARCC1 requires careful optimization based on tissue type and fixation method:

• Sample Preparation:

  • Formalin-fixed paraffin-embedded (FFPE) tissues should be sectioned at 4-5 μm thickness.

  • Fresh frozen tissues may yield better antigen preservation but require different fixation protocols.

• Antigen Retrieval Methods:

  • Heat-induced epitope retrieval (HIER) is essential for SMARCC1 detection in FFPE tissues.

  • Primary recommendation: TE buffer pH 9.0 provides optimal retrieval for most tissue types .

  • Alternative method: Citrate buffer pH 6.0 may be used, though effectiveness varies by tissue type .

  • Perform retrieval by heating sections in a pressure cooker or microwave for 15-20 minutes followed by gradual cooling.

• Antibody Dilution by Tissue Type:

  • For human malignant melanoma, appendicitis, brain, kidney, ovary, placenta, and testis tissues, dilutions between 1:250 and 1:1000 have been validated .

  • Optimal dilutions may vary between tissue types based on target abundance and tissue-specific characteristics.

  • Always perform a dilution series (e.g., 1:250, 1:500, 1:1000) during protocol optimization.

• Detection Systems:

  • HRP-polymer detection systems typically provide better sensitivity than avidin-biotin systems.

  • DAB (3,3'-diaminobenzidine) substrate provides good contrast for nuclear SMARCC1 staining.

  • Counterstain with hematoxylin should be optimized to avoid masking nuclear SMARCC1 staining.

• Controls and Validation:

  • Include known positive tissues (brain, testis) in each staining run.

  • Use isotype controls to assess non-specific binding.

  • Consider dual-staining with other SWI/SNF complex members to validate specificity.

By systematically optimizing these parameters for each tissue type, researchers can achieve consistent and specific SMARCC1 detection in immunohistochemical applications.

How is SMARCC1 dysregulation implicated in cancer development and progression?

SMARCC1 exhibits context-dependent roles in cancer biology, functioning as either a tumor suppressor or oncogene depending on the cancer type:

In prostate cancer (PCa), SMARCC1 predominantly acts as a tumor suppressor. Experimental knockdown of SMARCC1 in PCa cell lines significantly enhances cellular proliferation and viability. Colony formation assays demonstrate increased clonogenic capacity in SMARCC1-depleted cells . Cell cycle analysis reveals that SMARCC1 knockdown decreases the proportion of cells in G0/G1 phase while increasing S-phase populations, suggesting impaired cell cycle checkpoint control . At the molecular level, SMARCC1 suppresses PCa progression by inhibiting the PI3K/AKT signaling pathway, a key oncogenic driver .

Conversely, in bladder cancer (BC), SMARCC1 demonstrates oncogenic characteristics. RT-qPCR and western blot analyses confirm SMARCC1 upregulation in BC tissues and cell lines . Functional studies show that SMARCC1 knockdown inhibits cell proliferation, induces G0/G1 cell cycle arrest, promotes apoptosis, and reduces migration capacity in BC cells .

This dual nature of SMARCC1 highlights the complexity of chromatin remodeling factors in cancer and underscores the importance of context-specific investigation when considering SMARCC1 as a therapeutic target.

What role does SMARCC1 play in epithelial-mesenchymal transition (EMT) and metastasis?

SMARCC1's influence on epithelial-mesenchymal transition (EMT) and metastasis has been extensively characterized, particularly in prostate cancer models:

In functional studies, SMARCC1 knockdown significantly enhances the migratory capacity of prostate cancer cells. Transwell and wound healing assays demonstrate that loss of SMARCC1 substantially increases cancer cell migration in vitro . This phenotypic change is accompanied by dramatic alterations in EMT marker expression. SMARCC1-depleted cells exhibit marked increases in mesenchymal markers including vimentin and N-cadherin, with concurrent decreases in epithelial markers such as E-cadherin . Furthermore, SMARCC1 knockdown upregulates key EMT-related transcription factors, including Slug, Snail, and Zeb1, which are master regulators of the EMT program .

The in vivo significance of these findings has been validated using lung metastasis models. When SMARCC1-knockdown prostate cancer cells are injected, they demonstrate significantly enhanced metastatic potential compared to control cells . Histological analysis of lung metastatic nodules confirms both their prostate origin (through P504S staining) and altered expression of proteins involved in invasion and metastasis, including increased MMP2 expression and decreased epithelial junction proteins E-cadherin and claudin1 .

The molecular mechanisms underlying SMARCC1's regulation of EMT involve its role as part of the SWI/SNF chromatin remodeling complex, which modulates the accessibility of EMT-related gene promoters to transcription factors. This epigenetic regulation represents a critical layer of control over the metastatic cascade and highlights SMARCC1 as a potential therapeutic target for preventing cancer progression.

What are common challenges when using SMARCC1 antibodies for immunoprecipitation?

Researchers frequently encounter specific challenges when performing immunoprecipitation (IP) with SMARCC1 antibodies:

• High Molecular Weight Detection Issues:

  • SMARCC1 is a large protein (~155 kDa) that can be difficult to efficiently extract and detect.

  • Solution: Use low-percentage SDS-PAGE gels (6-8%) for better resolution of high molecular weight proteins and extend transfer times during Western blot confirmation.

• Complex Formation Interference:

  • SMARCC1 exists in multi-protein SWI/SNF complexes that may mask epitopes or interfere with antibody binding.

  • Solution: Consider using different lysis buffers with varying detergent strengths. For mouse brain tissue, successful IP has been documented using 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate .

• Cross-Reactivity Concerns:

  • Some antibodies may cross-react with other SWI/SNF complex members with similar structures.

  • Solution: Validate specificity using SMARCC1 knockout/knockdown controls and confirm results with multiple antibody clones if possible.

• Co-IP Partner Detection:

  • When performing co-immunoprecipitation to identify SMARCC1 interacting partners, weak or transient interactions may be lost during washing steps.

  • Solution: Consider crosslinking approaches or less stringent washing conditions to preserve weak interactions.

• Non-specific Background:

  • High background can obscure specific SMARCC1 signals in IP experiments.

  • Solution: Pre-clear lysates with protein A/G beads before antibody addition, use specific blocking agents, and optimize antibody amounts carefully.

By addressing these common challenges with the suggested solutions, researchers can improve the specificity and sensitivity of SMARCC1 immunoprecipitation experiments.

How can inconsistent results in SMARCC1 expression studies be reconciled?

When confronted with contradictory findings regarding SMARCC1 expression and function across different studies, consider these methodological and biological factors:

By systematically addressing these variables, researchers can better understand the complex and sometimes contradictory roles of SMARCC1 across different experimental systems and disease contexts.

What are the emerging therapeutic implications of targeting SMARCC1 in cancer?

SMARCC1's complex role in cancer biology presents both challenges and opportunities for therapeutic development. The context-dependent function of SMARCC1 necessitates cancer type-specific approaches to targeting this protein. In cancers where SMARCC1 demonstrates tumor-suppressive properties, such as clear cell renal cell carcinoma and prostate cancer, therapeutic strategies might focus on restoring or enhancing SMARCC1 expression or activity . Conversely, in contexts where SMARCC1 exhibits oncogenic properties, like bladder cancer, direct inhibition strategies may prove beneficial .

The nuclear transport machinery regulating SMARCC1 localization presents an intriguing therapeutic avenue. Research has identified KPNA2, Nup50, and Nup153 as key regulators of SMARCC1 nuclear translocation in bladder cancer . Targeting these transport mechanisms could provide a novel approach to modulating SMARCC1 activity, particularly in cancers where its nuclear function drives disease progression.