SMARCC2 Antibody, Biotin conjugated

What is SMARCC2 and why is it significant in chromatin remodeling research?

SMARCC2, also known as BAF170, is a critical subunit of the SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complex. This protein functions as a core component that interacts with other complex members including SMARCA4 (BRG1), SMARCC1 (BAF155), and SMARCB1 (BAF47) to regulate chromatin accessibility and transcriptional processes . The SWI/SNF complex plays essential roles in modifying nucleosome positioning through ATP-dependent mechanisms, thereby influencing diverse cellular processes including development, differentiation, and disease pathogenesis. Understanding SMARCC2 function has significant implications for both fundamental chromatin biology and disease mechanisms, particularly in developmental disorders and cancer biology contexts where SWI/SNF complex dysfunction has been implicated .

What are the key specifications to consider when selecting a biotin-conjugated SMARCC2 antibody?

When selecting a biotin-conjugated SMARCC2 antibody for research applications, multiple technical specifications must be evaluated to ensure experimental success:

Researchers should select antibodies with validation data that aligns with their intended application and experimental system, considering that reactivity and performance may vary significantly between applications .

Why use a biotin-conjugated SMARCC2 antibody instead of unconjugated versions?

Biotin-conjugated SMARCC2 antibodies offer several methodological advantages over unconjugated alternatives:

• Enhanced detection sensitivity: The biotin-streptavidin system provides signal amplification through the high-affinity binding interaction (Kd ≈ 10^-15 M), which can significantly improve detection limits in various assays .

• Versatile visualization options: A single biotin-conjugated primary antibody can be used with multiple streptavidin-conjugated detection systems (fluorophores, enzymes, quantum dots), providing experimental flexibility without requiring multiple specialized secondary antibodies .

• Reduced background interference: The biotin-conjugation can eliminate secondary antibody cross-reactivity issues that often plague multi-species co-localization studies, making it particularly valuable for complex immunostaining protocols .

• Compatibility with streptavidin-based enrichment: Biotin-conjugated antibodies facilitate efficient protein complex isolation in pull-down or proximity labeling experiments, similar to the TurboID approaches used with related SWI/SNF complex proteins .

How can SMARCC2 antibodies be optimized for investigating chromatin remodeling mechanisms?

Investigating chromatin remodeling mechanisms using SMARCC2 antibodies requires careful optimization:

For chromatin immunoprecipitation (ChIP) applications, researchers should implement the following optimization strategies:

• Cross-linking optimization: Test both formaldehyde concentrations (0.1-1%) and incubation times (5-15 minutes) to balance efficient protein-DNA cross-linking while maintaining epitope accessibility. The extended structure of SMARCC2 within the SWI/SNF complex may require gentler cross-linking conditions.

• Sonication parameters: Optimize sonication conditions to generate 200-500bp DNA fragments while maintaining SMARCC2 epitope integrity, as excessive sonication can destroy antigenic determinants.

• Antibody validation: Confirm SMARCC2 antibody specificity prior to ChIP applications through Western blotting and immunoprecipitation, as demonstrated with various SMARCC2 antibodies targeting specific amino acid regions (e.g., AA 300-647, AA 361-410) .

• Controls implementation: Include appropriate controls such as IgG negative controls and positive controls targeting known SMARCC2-associated gene regions, particularly those identified in SWI/SNF complex binding studies .

• Sequential ChIP approach: For examining SMARCC2 co-occupancy with other SWI/SNF components, sequential ChIP approaches can determine whether SMARCC2 simultaneously occupies chromatin with other factors such as SMARCB1 or transcription factors like JUN that have been shown to interact with SWI/SNF complexes .

What methodological approaches can be used to investigate SMARCC2 interactions with other SWI/SNF complex members?

Several sophisticated methodological approaches can be employed to characterize SMARCC2 interactions:

• Co-immunoprecipitation with biotin-conjugated antibodies: Utilizing biotin-conjugated SMARCC2 antibodies for co-IP allows for efficient pull-down of protein complexes using streptavidin beads. This approach has been effective in demonstrating that wild-type SMARCC2 co-precipitates with SMARCA4 (BRG1), SMARCC1 (BAF155), and SMARCB1 (BAF47), while mutant versions fail to maintain these interactions .

• Proximity labeling techniques: Adapting approaches similar to those used with TurboID-SMARCD1 for SMARCC2 allows for temporal mapping of protein interactions. This technique can identify both stable and transient interactions within the SWI/SNF complex during assembly or in response to cellular signals .

• Mass spectrometry analysis: Following SMARCC2 immunoprecipitation, tandem mass spectrometry (MS/MS) can identify novel interaction partners and post-translational modifications that regulate complex assembly and function, similar to the TMT-MS approach used with related SWI/SNF components .

• FRET/BRET assays: For studying dynamic interactions in living cells, fluorescence or bioluminescence resonance energy transfer can be employed by tagging SMARCC2 and potential interacting partners with appropriate donor/acceptor pairs.

• Mutational analysis: Strategic mutations in SMARCC2 can help map specific interaction domains, as demonstrated by experiments showing that mutant versions of related SWI/SNF proteins fail to co-precipitate with core complex members .

How do researchers distinguish between different SWI/SNF complex assemblies containing SMARCC2?

Distinguishing between different SWI/SNF complex assemblies requires sophisticated methodological approaches:

• Differential co-immunoprecipitation: Biotin-conjugated SMARCC2 antibodies can be used to pull down complex members, followed by western blotting for subunits specific to different SWI/SNF subcomplexes (BAF vs. PBAF). Quantitative analysis of co-precipitated proteins can reveal the distribution of SMARCC2 among different complexes .

• Density gradient ultracentrifugation: This technique separates protein complexes based on size and shape, allowing isolation of distinct SWI/SNF assemblies containing SMARCC2 for further analysis.

• Glycerol gradient sedimentation: This approach can separate different-sized complexes containing SMARCC2, followed by western blotting to identify subcomplex-specific components.

• Chromatin targeting analysis: CUT&RUN or ChIP-seq approaches can map genome-wide binding profiles of SMARCC2 in comparison with subcomplex-specific components, revealing distinct targeting patterns associated with different SWI/SNF assemblies .

• Functional genomics approaches: Comparing the transcriptional consequences of SMARCC2 depletion versus depletion of subcomplex-specific components can help determine which functions are associated with specific SWI/SNF assemblies.

What are common issues when using biotin-conjugated SMARCC2 antibodies in Western blotting and how can they be resolved?

When troubleshooting, it's important to note the significant discrepancy between the calculated molecular weight of SMARCC2 (132.9 kDa) and some observed weights in experimental systems (39 kDa) , which may indicate specific isoforms, cleavage products, or technical artifacts that require careful validation.

How can researchers optimize ELISA protocols using biotin-conjugated SMARCC2 antibodies?

Optimizing ELISA protocols with biotin-conjugated SMARCC2 antibodies requires systematic adjustment of multiple parameters:

• Coating optimization: When using direct ELISA for SMARCC2 detection, optimize coating buffer pH (typically pH 7.4-9.6) and concentration of capture antibody or target protein.

• Blocking optimization: Test different blocking agents (BSA, non-fat milk, commercial blockers) at various concentrations (1-5%) to minimize background while maintaining specific signal. For biotin-conjugated antibodies, use biotin-free blocking reagents to avoid interference.

• Antibody dilution series: Perform a titration of the biotin-conjugated SMARCC2 antibody to determine optimal concentration, starting with the manufacturer's recommended range (typically within 1:500-1:2000) .

• Detection system calibration: For streptavidin-conjugated detection enzymes, optimize concentration and incubation time to maximize signal-to-noise ratio.

• Cross-reactivity assessment: Validate specificity by including controls with related proteins (SMARCD1, SMARCD2) to ensure the antibody specifically recognizes SMARCC2.

• Sample preparation considerations: Nuclear proteins like SMARCC2 may require special extraction protocols with nuclear lysis buffers containing appropriate detergents (0.1-1% NP-40 or Triton X-100) to maintain protein structure while ensuring efficient extraction.

What controls should be included when using SMARCC2 antibodies in chromatin immunoprecipitation studies?

Comprehensive controls are essential for valid chromatin immunoprecipitation studies with SMARCC2 antibodies:

• Input control: Retain 5-10% of pre-immunoprecipitation chromatin to normalize precipitation efficiency.

• Isotype control: Include matched isotype IgG from the same species as the SMARCC2 antibody (rabbit IgG for most commercially available options) to assess non-specific binding.

• Positive genomic controls: Include PCR primers for regions known to be bound by SWI/SNF complexes, such as promoters of genes regulated by this complex based on previous studies .

• Negative genomic controls: Include primers for genomic regions not expected to be bound by SWI/SNF (e.g., gene deserts or unexpressed genes in your cell system).

• Cell type controls: When possible, include both cell types expressing SMARCC2 and those with reduced or no expression as biological controls.

• Knockdown/knockout validation: If available, include samples from SMARCC2-depleted cells to confirm antibody specificity.

• Sequential ChIP controls: For co-occupancy studies, perform reverse-order sequential ChIP to confirm results (e.g., SMARCC2→SMARCB1 and SMARCB1→SMARCC2) to validate true co-occupancy rather than separate binding events .

How can biotin-conjugated SMARCC2 antibodies be used to investigate the role of SWI/SNF complex in disease models?

Biotin-conjugated SMARCC2 antibodies provide valuable tools for investigating SWI/SNF complex dysregulation in disease:

• Cancer research applications: The SWI/SNF complex has tumor suppressor functions, with alterations in complex members implicated in various malignancies. Biotin-conjugated SMARCC2 antibodies can be used to analyze complex composition and chromatin targeting in cancer cells, particularly in leukemia models where SWI/SNF components like SMARCD2 have been shown to regulate myeloid differentiation .

• Developmental disorder investigations: SWI/SNF complex members regulate developmental processes, and mutations can cause congenital abnormalities. Biotin-conjugated SMARCC2 antibodies can help characterize how mutations affect complex assembly and function in relevant cell models .

• Mechanistic studies in patient-derived cells: In studies of neutropenia and myelodysplasia associated with SMARCD2 mutations, biotin-conjugated SMARCC2 antibodies can be used to examine how related complex members are affected by these mutations .

• Drug screening applications: For compounds targeting SWI/SNF complex activity, biotin-conjugated SMARCC2 antibodies can serve as tools to monitor drug effects on complex assembly and chromatin targeting.

• Post-translational modification analysis: Biotin-conjugated antibodies can facilitate enrichment of SMARCC2 for analysis of post-translational modifications that may be altered in disease states.

What considerations are important when analyzing co-localization data involving SMARCC2?

When analyzing SMARCC2 co-localization with other proteins, several technical and biological factors require careful consideration:

• Resolution limitations: Standard confocal microscopy has a resolution limit (~200nm) that may falsely suggest co-localization of proteins that are not directly interacting. Super-resolution techniques should be considered for definitive co-localization studies.

• Antibody validation: Confirm the specificity of both the biotin-conjugated SMARCC2 antibody and antibodies against potential interaction partners through appropriate controls, including single-antibody controls and blocking peptides where available .

• Quantitative co-localization metrics: Apply appropriate statistical measures (Pearson's correlation coefficient, Manders' overlap coefficient) rather than relying on visual assessment alone.

• Biological context interpretation: Consider that SMARCC2 participates in different SWI/SNF subcomplexes, so co-localization patterns may vary depending on cell type, differentiation state, or disease context .

• Dynamic interaction considerations: SMARCC2 interactions may be transient or regulated by cellular signals, requiring time-resolved approaches similar to those used with other SWI/SNF components .

• Nuclear compartmentalization effects: As a nuclear protein involved in chromatin remodeling, SMARCC2 localization is influenced by chromatin states and nuclear architecture, which should be considered when interpreting apparent co-localization patterns.

How might emerging proximity labeling technologies enhance SMARCC2 interaction studies?

Emerging proximity labeling approaches offer exciting opportunities for SMARCC2 research:

• TurboID and miniTurbo applications: Adapting approaches used with SMARCD1 to SMARCC2 could provide temporal resolution of protein interactions during complex assembly or cellular transitions. This approach could identify both stable core interactions and transient regulatory interactions that might be missed by traditional co-immunoprecipitation.

• Split-BioID systems: These could be applied to study specific SMARCC2 domain interactions with particular partners, providing spatial resolution of interaction interfaces within the complex architecture.

• APEX2-based proximity labeling: This rapid labeling approach could capture extremely transient interactions of SMARCC2 with transcription factors or chromatin regions during dynamic processes like transcriptional activation.

• Multiplexed proximity labeling: Combining different orthogonal proximity labeling enzymes could allow simultaneous mapping of multiple SWI/SNF component interactomes, including SMARCC2, providing a systems-level view of complex assembly and function.

• In vivo applications: Adapting these technologies to model organisms could reveal tissue-specific or developmental stage-specific SMARCC2 interactions relevant to its biological functions in differentiation and development .

These approaches would complement existing co-immunoprecipitation studies that have already revealed important insights about SMARCC2 interactions with core SWI/SNF components like SMARCA4, SMARCC1, and SMARCB1 .