SMARCC2 Antibody

What is SMARCC2/BAF170 and what is its role in chromatin remodeling?

SMARCC2/BAF170 is one of the core subunits of the SWI/SNF (Switch/Sucrose Non-Fermentable) complex, which plays a critical role in nucleosome remodeling. This protein is necessary for efficient nucleosome remodeling by Brg1 in vitro and contributes to chromatin structure regulation. SMARCC2/BAF170 is predominantly found in non-pluripotent cells and is notably absent in pluripotent embryonic stem (ES) cells. The protein is part of the BAF (Brg/Brahma-Associated Factor) complex variant of SWI/SNF and has been implicated in developmental processes and cellular differentiation pathways .

During neuronal differentiation, SMARCC2/BAF170 expression is upregulated following retinoic acid treatment of mouse ES cells. Interestingly, exogenous expression of SMARCC2/BAF170 has been shown to trigger loss of stem cell pluripotency and self-renewal capabilities, indicating its essential role in developmental transitions . Recent studies have also linked germline variants in SMARCC2 to neurodevelopmental disorders, collectively termed "BAFopathies," which include conditions similar to Coffin-Siris and Nicolaides-Baraitser syndromes .

What applications are most effective for SMARCC2 antibodies in research?

SMARCC2 antibodies have demonstrated utility across multiple experimental applications, with varying degrees of effectiveness based on the specific antibody clone and experimental conditions:

When selecting applications, researchers should consider that Western blotting appears to be the most consistently validated application across multiple antibody suppliers. For detecting endogenous SMARCC2, sensitivity levels and detection thresholds will vary between antibodies, making validation in your specific experimental system critical .

What molecular characteristics of SMARCC2 should researchers consider when selecting antibodies?

When selecting SMARCC2 antibodies, researchers should consider several key molecular characteristics:

• Molecular Weight: SMARCC2 protein typically appears at 162-170 kDa on Western blots, though some literature reports it at 132.9 kilodaltons. This variability may reflect post-translational modifications or alternative splicing .

• Species Reactivity: Most commercially available antibodies demonstrate reactivity with human SMARCC2, with many also cross-reacting with mouse and rat orthologs. Some antibodies may also react with monkey samples. Researchers should verify species cross-reactivity based on the high sequence homology (often 100%) between human SMARCC2 and orthologs in experimental model organisms .

• Epitope Location: Consider whether the antibody targets regions that might be affected by known variants or post-translational modifications. This is particularly important for studies examining specific SMARCC2 variants associated with neurodevelopmental disorders .

• Alternative Names: When searching literature and resources, be aware that SMARCC2 may also be referred to as BAF170, CRACC2, CSS8, Rsc8, SWI/SNF complex subunit SMARCC2, or SWI/SNF complex 170 kDa subunit .

How can researchers optimize Western blot protocols for detecting endogenous SMARCC2 in different cell types?

Optimizing Western blot protocols for SMARCC2 detection requires attention to several critical parameters:

• Sample Preparation: For efficient extraction of nuclear proteins like SMARCC2, use nuclear extraction protocols that include DNase treatment to release chromatin-bound proteins. Standard RIPA buffer may be insufficient for complete extraction of nuclear matrix-associated proteins.

• Protein Loading: Load 20-40 μg of total protein per lane, with higher amounts potentially needed for cell types with lower SMARCC2 expression. Nuclear extracts typically require less total protein (10-20 μg) for equivalent signal.

• Gel Percentage and Running Conditions: Due to the high molecular weight of SMARCC2 (162-170 kDa), use 6-8% polyacrylamide gels or gradient gels (4-15%). Extend running time at lower voltages (80-100V) to improve resolution of high-molecular-weight proteins .

• Transfer Conditions: For efficient transfer of high-molecular-weight proteins, use wet transfer systems with 10-20% methanol and longer transfer times (90-120 minutes) at constant amperage. Consider adding SDS (0.1%) to the transfer buffer to facilitate the movement of large proteins.

• Antibody Dilution and Incubation: Most SMARCC2 antibodies work optimally at 1:1000 dilution for Western blotting. Overnight incubation at 4°C typically yields better results than shorter incubations at room temperature .

• Cell-Type Specific Considerations: Expression levels vary significantly between cell types, with pluripotent stem cells showing minimal expression compared to differentiated cells, particularly neuronal lineages. Adjust protein loading and exposure times accordingly .

What approaches can effectively distinguish between different SMARCC2 variants in research on BAFopathies?

Effectively distinguishing between SMARCC2 variants associated with BAFopathies requires a multi-faceted approach:

• Variant-Specific Antibodies: While commercially available antibodies typically recognize wild-type SMARCC2, researchers studying specific variants may need to develop custom antibodies targeting variant-specific epitopes or use epitope-tagged constructs for overexpression studies .

• Functional Assays: Different SMARCC2 variants can be characterized by their impact on BAF complex assembly and function. Combining techniques such as co-immunoprecipitation (CoIP) and proximity ligation assays (PLA) can reveal how variants affect interactions with other BAF subunits such as ARID1B, SMARCA4, SMARCC1, and SMARCE1 .

• Cellular Localization Analysis: While most missense variants of SMARCC2 exhibit normal nuclear localization, subtle differences in subnuclear distribution or chromatin association can be detected using high-resolution immunofluorescence or chromatin fractionation followed by Western blotting .

• Expression Level Analysis: Real-time PCR can be used to measure SMARCC2 expression levels, particularly important for variants affecting transcription or mRNA stability. This approach has been demonstrated effective for variants like c.1311-3C>G, which reduces SMARCC2 expression .

• Genotype-Phenotype Correlation: Clinical studies have begun to identify distinct phenotypic patterns associated with likely gene-disrupting (LGD) variants versus non-truncating variants of SMARCC2, providing context for laboratory findings .

How can SMARCC2 antibodies be utilized to study BAF complex dynamics during cellular differentiation?

Studying BAF complex dynamics during cellular differentiation requires sophisticated applications of SMARCC2 antibodies:

• Temporal Expression Analysis: Western blotting with SMARCC2 antibodies at different time points during differentiation can track the upregulation of SMARCC2/BAF170, particularly in neuronal differentiation models. This is especially relevant given that SMARCC2 expression increases during retinoic acid-induced differentiation of mouse ES cells .

• Chromatin Immunoprecipitation Sequencing (ChIP-seq): SMARCC2 antibodies can be used for ChIP-seq to map genome-wide binding patterns during differentiation, revealing how BAF complex localization changes as cells exit pluripotency and adopt lineage-specific fates.

• BAF Complex Composition Analysis: Co-immunoprecipitation with SMARCC2 antibodies followed by mass spectrometry can identify differentiation-specific changes in BAF complex composition and subunit switching. This is particularly important as SMARCC2 is notably absent in pluripotent cells but becomes incorporated into the complex during differentiation .

• Single-Cell Analyses: Combining SMARCC2 antibodies with other BAF subunit antibodies in multi-parameter flow cytometry or single-cell Western blotting can reveal heterogeneity in BAF complex composition within differentiating populations.

• Super-Resolution Microscopy: Using fluorescently labeled SMARCC2 antibodies in super-resolution microscopy techniques allows for visualization of BAF complex assembly and chromatin association during key differentiation transitions at the nanoscale level.

What validation methods should researchers employ to confirm SMARCC2 antibody specificity?

Rigorous validation of SMARCC2 antibodies is essential for reliable research outcomes:

• Knockout/Knockdown Controls: The gold standard for antibody validation is testing in SMARCC2 knockout or knockdown systems. Western blots should show absence or significant reduction of the band at 162-170 kDa in these negative controls.

• Overexpression Systems: Testing antibodies in systems overexpressing tagged SMARCC2 can confirm the ability to detect increased protein levels and verify the molecular weight.

• Peptide Competition Assays: Pre-incubating the antibody with the immunizing peptide should abolish or significantly reduce signal in Western blot, immunofluorescence, or immunohistochemistry applications.

• Cross-Reactivity Assessment: Test antibodies across multiple species based on sequence homology. For example, antibodies designed against human SMARCC2 may cross-react with mouse and rat orthologs due to high sequence conservation .

• Multiple Antibody Comparison: Using different antibodies targeting distinct epitopes of SMARCC2 should yield consistent results in terms of molecular weight, subcellular localization, and expression patterns.

• Application-Specific Validation: For specialized applications like proximity ligation assays or chromatin immunoprecipitation, additional controls should confirm that antibodies can access epitopes in their native conformational state within protein complexes .

What methodological approaches can resolve contradictory findings when using SMARCC2 antibodies in chromatin studies?

When researchers encounter contradictory findings using SMARCC2 antibodies in chromatin studies, several methodological approaches can help resolve discrepancies:

• Epitope Accessibility Analysis: Different fixation and extraction methods can significantly impact epitope accessibility, particularly for chromatin-bound proteins. Testing multiple fixation protocols (formaldehyde, methanol, or combinations) can determine optimal conditions for specific antibodies.

• Chromatin State Considerations: The condensation state of chromatin can affect antibody access to SMARCC2 within the BAF complex. Implementing chromatin relaxation steps (like brief nuclease treatment) before antibody application may improve detection in compact heterochromatin regions.

• BAF Complex Configuration Effects: SMARCC2 exists in different BAF complex configurations that may mask certain epitopes. Using antibodies targeting different regions of SMARCC2 can help distinguish whether contradictory results stem from differential epitope accessibility in various complex assemblies.

• Quantitative Method Standardization: Implementing strictly standardized quantitative Western blot protocols with titrated loading controls can address inconsistencies in protein level measurements. This includes using purified recombinant SMARCC2 for standard curves when absolute quantification is needed.

• Cell Type-Specific Control Panels: Creating comprehensive control panels of cell types with well-characterized SMARCC2 expression levels (from high expression in neuronal lineages to negligible expression in pluripotent stem cells) provides essential reference points for antibody performance evaluation across experimental conditions .