SMARCA2 Antibody

What is SMARCA2 and why is it important in research?

SMARCA2, also known as BRM (brahma), is a critical catalytic subunit of the SWI/SNF family of ATP-dependent chromatin remodeling complexes. It functions by utilizing energy from ATP hydrolysis to alter chromatin structure and activate transcription of select genes. SMARCA2 is involved in both transcriptional activation and repression through its ability to change DNA-nucleosome topology . As a component of both neural progenitor-specific chromatin remodeling complex (npBAF) and neuron-specific chromatin remodeling complex (nBAF), SMARCA2 plays crucial roles in neural development, particularly in the transition from proliferating neural stem/progenitor cells to postmitotic neurons . The importance of SMARCA2 extends to synthetic lethality contexts in certain cancers, particularly where its paralog SMARCA4 is mutated, making it an attractive therapeutic target .

What applications are SMARCA2 antibodies validated for?

SMARCA2 antibodies have been validated for multiple research applications including:

• Chromatin Immunoprecipitation (ChIP): Using approximately 10 μg per ChIP reaction, allowing researchers to study SMARCA2 binding to specific genomic regions

• Western Blotting (WB): For detecting SMARCA2 protein expression levels in cell and tissue lysates

• Immunocytochemistry/Immunofluorescence (ICC/IF): Typically at 0.5 μg/ml dilution to visualize SMARCA2 subcellular localization

• Immunoprecipitation (IP): For isolating SMARCA2 protein complexes to study interaction partners

The choice of application determines the optimal antibody format and experimental conditions. For chromatin studies, antibodies validated for ChIP and ChIP-Seq are essential for reliable chromatin-binding data .

How do I select the appropriate SMARCA2 antibody for my experiment?

When selecting a SMARCA2 antibody, consider these critical factors:

• Antibody type: Monoclonal antibodies (such as clone 1H7A10) offer high specificity and reproducibility, while polyclonal antibodies may provide broader epitope recognition

• Species reactivity: Confirm the antibody's reactivity with your experimental species. Many SMARCA2 antibodies are validated for human samples, but cross-reactivity with mouse or other species should be verified

• Application validation: Ensure the antibody has been specifically validated for your intended application (ChIP, WB, IF, etc.)

• Epitope location: Consider the epitope region recognized by the antibody. For example, some antibodies target regions corresponding to amino acids 48-214 of mouse BRM or within amino acids 1300-1400 of human SMARCA2

• Positive controls: Use appropriate positive controls, such as HeLa nuclear extract, which can serve as a positive control for SMARCA2 detection

The selection criteria should align with your specific experimental goals and model system to ensure optimal results.

What is the difference between SMARCA2 (BRM) and SMARCA4 (BRG1)?

SMARCA2 (BRM) and SMARCA4 (BRG1) are paralogous catalytic subunits of the SWI/SNF chromatin remodeling complex with significant structural similarities:

This high degree of homology presents challenges for developing selective targeting agents, particularly in therapeutic contexts where distinguishing between these paralogs is crucial . When selecting antibodies, researchers should carefully verify specificity between these closely related proteins.

How can SMARCA2 antibodies be utilized in chromatin remodeling complex studies?

SMARCA2 antibodies serve as powerful tools for investigating the composition and function of SWI/SNF chromatin remodeling complexes through several advanced approaches:

• ChIP-Seq analysis: SMARCA2 antibodies can be used with ChIP-Seq to map genome-wide binding patterns, revealing target genes and regulatory elements. This approach requires highly specific antibodies validated for ChIP applications, such as those compatible with ChIP-IT High Sensitivity Kit or magnetic bead-based ChIP-IT Express Kits .

• Co-immunoprecipitation (Co-IP): IP-grade SMARCA2 antibodies can pull down associated complex components, enabling researchers to identify context-specific interaction partners and complex assemblies .

• Sequential ChIP (Re-ChIP): This technique uses antibodies against SMARCA2 and other chromatin regulators sequentially to determine co-occupancy at specific genomic loci, providing insights into combinatorial regulation.

• CUT&RUN/CUT&Tag approaches: These newer techniques offer higher resolution and sensitivity than traditional ChIP, requiring less starting material and providing cleaner background for SMARCA2 genomic mapping .

The methodological sophistication of these approaches requires careful antibody validation and optimization to ensure that the recognized epitope remains accessible in the context of the assembled chromatin remodeling complex.

What roles does SMARCA2 play in antiviral immunity and how can antibodies help investigate this function?

SMARCA2 has emerged as a critical factor in antiviral immunity, particularly against influenza viruses, making it an important subject for immunological research:

• Regulation of interferon-stimulated genes (ISGs): SMARCA2 is essential for the expression of certain ISGs and establishment of an antiviral state. SMARCA2 antibodies can be used in ChIP experiments to identify which ISG promoters are directly regulated by SMARCA2-containing complexes .

• MxA-mediated antiviral activity: Interestingly, while SMARCA2 is required for the antiviral activity of MxA against influenza viruses (particularly H5N1 and H7N7), it's not needed for MxA expression itself. Rather, SMARCA2 regulates factors that enable MxA's antiviral function, including IFITM2 and IGFBP3 .

• Experimental verification: RNAi-mediated depletion of SMARCA2 in A549-MxA cells resulted in >1 log₁₀ increase in viral titer of H5N1 strain A/Thailand/1(KAN-1)/2004, demonstrating its functional importance .

Methodologically, researchers can combine SMARCA2 antibodies with virus infection studies, analyzing changes in chromatin occupancy during infection through ChIP-Seq and correlating these changes with transcriptional outcomes via RNA-Seq.

How can SMARCA2 antibodies be used to study synthetic lethality in cancer research?

The synthetic lethal relationship between SMARCA2 and SMARCA4 presents significant research and therapeutic opportunities in cancer biology:

• Identification of SMARCA4-mutant contexts: SMARCA2 antibodies can be used in immunohistochemistry and western blotting to characterize tumor samples for SMARCA2 dependency, particularly in contexts where SMARCA4 is mutated or inactivated .

• Validation of PROTAC efficacy: In research involving proteolysis-targeting chimeras (PROTACs) like A947, which selectively degrade SMARCA2, antibodies are essential for confirming target degradation and selectivity over SMARCA4. Western blotting with specific antibodies provides quantitative assessment of degradation efficiency .

• Mechanistic studies: ChIP-Seq with SMARCA2 antibodies before and after PROTAC treatment can reveal genomic loci that lose SMARCA2 occupancy, connecting chromatin changes to downstream transcriptional effects and cellular phenotypes .

• Resistance mechanisms: In models developing resistance to SMARCA2 targeting, antibodies help characterize compensatory mechanisms through immunoprecipitation coupled with mass spectrometry to identify altered protein interactions .

This research area exemplifies how SMARCA2 antibodies bridge basic chromatin biology with translational cancer research, enabling both mechanistic insights and therapeutic development strategies.

What are the critical factors for successful ChIP experiments with SMARCA2 antibodies?

Chromatin immunoprecipitation with SMARCA2 antibodies requires careful optimization for successful outcomes:

• Antibody amount optimization: Typically, 10 μg of SMARCA2 antibody per ChIP reaction is recommended, but this should be titrated for each experimental system . Insufficient antibody can lead to weak signals, while excess antibody may increase background.

• Chromatin preparation: SMARCA2 predominantly binds open chromatin regions, requiring optimized sonication conditions to properly fragment chromatin (typically 200-500 bp fragments). Over-sonication can destroy epitopes, while under-sonication reduces antibody accessibility.

• Fixation conditions: Standard 1% formaldehyde for 10 minutes works for many experiments, but optimization may be necessary. Overfixation can mask epitopes, while underfixation may not preserve protein-DNA interactions.

• Controls: Include:

  • Input chromatin (non-immunoprecipitated)

  • Negative control antibody (IgG matched to host species)

  • Positive control antibody targeting abundant chromatin proteins

  • Known SMARCA2-bound regions as positive control loci

• Washing stringency: Balance between removing non-specific interactions and preserving specific ones. Gradually increasing salt concentration in wash buffers often helps optimize signal-to-noise ratio.

For challenging samples or limited material, consider specialized approaches like ChIP-IT High Sensitivity or magnetic bead-based ChIP-IT Express Kits that have been validated with SMARCA2 antibodies .

How do I troubleshoot weak or nonspecific signals when using SMARCA2 antibodies?

When encountering weak or nonspecific signals with SMARCA2 antibodies, consider these methodological solutions:

For Western Blotting:

• Antibody concentration: Try different dilutions; recommended starting dilution may require adjustment based on expression levels in your sample

• Blocking optimization: Test alternative blocking agents (BSA vs. milk) as some epitopes may be masked by certain blockers

• Sample preparation: Ensure nuclear extraction is efficient, as SMARCA2 is predominantly nuclear and may be underrepresented in whole cell lysates

• Positive controls: Include HeLa nuclear extract as a positive control for SMARCA2 detection

For Immunofluorescence:

• Fixation method: Compare paraformaldehyde, methanol, and acetone fixation as epitope accessibility may differ

• Antigen retrieval: Implement citrate buffer or alternative antigen retrieval methods if working with fixed tissues

• Antibody concentration: Start with recommended 0.5 μg/ml dilution but adjust if needed

• Detection system: Enhanced detection systems may help with weak signals

For ChIP:

• Chromatin quality: Ensure proper sonication and check DNA fragment size

• Antibody batch: Different lots may have varying performance; request validation data specific to your lot

• Wash stringency: Adjust salt concentration in wash buffers to reduce background

• Crosslinking time: Optimize formaldehyde fixation duration and concentration

For applications with persistent difficulties, consider alternative antibody clones or epitopes that may provide better accessibility in your specific experimental context.

How should SMARCA2 antibodies be stored and handled to maintain optimal performance?

Proper storage and handling of SMARCA2 antibodies is critical for maintaining their performance over time:

• Storage temperature: Most SMARCA2 antibodies should be stored at -20°C for long-term storage. Some formulations containing glycerol (typically 30%) and small amounts of sodium azide (0.035%) can be stored at 4°C for short periods .

• Aliquoting: Upon receipt, divide the antibody into small working aliquots to minimize freeze-thaw cycles, which can degrade antibody quality. Each freeze-thaw cycle can reduce activity by approximately 10-15%.

• Thawing protocol: Thaw antibodies on ice or at 4°C rather than at room temperature to preserve protein structure and function.

• Working dilution preparation: Prepare working dilutions immediately before use and avoid storing diluted antibody solutions for extended periods.

• Buffer compatibility: When designing experiments, be aware that sodium azide in antibody storage buffers can inhibit HRP activity in certain applications and is toxic to living cells, necessitating thorough washing before cell-based assays .

• Transport conditions: For shipment between laboratories, use cold packs and insulated containers to maintain temperature.

• Contamination prevention: Use sterile techniques when handling antibody solutions to prevent microbial contamination, which can degrade antibodies.

Adherence to these storage and handling guidelines will help ensure consistent performance and reproducible results across experiments.

How do I interpret ChIP-Seq data generated with SMARCA2 antibodies?

Interpreting ChIP-Seq data generated with SMARCA2 antibodies requires understanding SMARCA2's biological context and several analytical considerations:

• Expected binding patterns: SMARCA2, as a chromatin remodeler, typically shows broad binding patterns rather than sharp peaks at regulatory elements. Analysis should use algorithms optimized for broad peak detection rather than standard peak callers designed for transcription factors.

• Genomic distribution analysis: SMARCA2 binding should be analyzed in relation to:

  • Promoter regions (typically within 2kb of transcription start sites)

  • Enhancers (often marked by H3K27ac)

  • Open chromatin regions (correlate with ATAC-seq data)

  • Cell-type specific regulatory elements

• Integration with transcriptomic data: Since SMARCA2 functions in transcriptional regulation, correlate binding sites with RNA-Seq data to identify genes directly regulated by SMARCA2-containing complexes, particularly in the context of the npBAF and nBAF complexes during neural development .

• Motif analysis: Perform de novo motif discovery on SMARCA2-bound regions to identify potential interaction partners, as SMARCA2 binds DNA non-specifically but is often recruited by sequence-specific transcription factors .

• Comparative analysis: Compare SMARCA2 binding patterns with:

  • SMARCA4 (BRG1) binding (paralog with similar function)

  • Other SWI/SNF components to identify complex integrity

  • Histone modifications indicative of active/repressed chromatin

• Pathway enrichment: Perform Gene Ontology and pathway analysis on SMARCA2-bound genes to identify biological processes under SMARCA2 regulation.

These analytical approaches help distinguish true binding events from artifacts and place SMARCA2 binding in its appropriate biological context.

How can I validate SMARCA2 antibody specificity for my experiments?

• Genetic validation:

  • Use SMARCA2 knockout or knockdown models (siRNA, shRNA, or CRISPR) to confirm signal loss

  • For ChIP applications, perform ChIP in SMARCA2-depleted cells to identify non-specific binding

  • In the context of H5N1 virus experiments, SMARCA2 knockdown should result in >1 log₁₀ increase in viral titer if the antibody is specific

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide (if using antibodies raised against amino acids 48-214 of mouse BRM or amino acids 1300-1400 of human SMARCA2)

  • Signal should be substantially reduced in the presence of competing peptide

• Paralog specificity:

  • Test antibody reactivity against both SMARCA2 and SMARCA4 in parallel

  • This is particularly important given their 93% ATPase domain identity and 96% bromodomain identity

  • In Western blots, distinguish based on slightly different molecular weights

• Multi-antibody validation:

  • Compare results using antibodies targeting different SMARCA2 epitopes

  • Consistent results with different antibodies increase confidence in specificity

• Mass spectrometry verification:

  • For immunoprecipitation experiments, confirm pulled-down protein identity by mass spectrometry

  • This approach can also identify non-specific binding partners

• Positive controls:

  • Include HeLa nuclear extract as a positive control for SMARCA2 detection

  • Use known SMARCA2-expressing tissues or cell lines appropriate for your experimental system

Thorough validation using multiple approaches provides the strongest evidence for antibody specificity, which is particularly important when studying closely related proteins like SMARCA2 and SMARCA4.

What controls should I include when using SMARCA2 antibodies in various experimental settings?

Proper experimental controls are essential when working with SMARCA2 antibodies to ensure valid interpretation of results:

For Western Blotting:

• Positive control: HeLa nuclear extract serves as a reliable positive control for SMARCA2 detection

• Loading control: Include nuclear protein controls like Lamin B1 or HDAC1

• Negative control: Include samples from SMARCA2 knockdown/knockout models

• Size marker: Use to confirm the expected molecular weight (~180-190 kDa)

For ChIP/ChIP-Seq:

• Input control: Non-immunoprecipitated chromatin (typically 1-5%)

• IgG control: Isotype-matched IgG from the same species as the SMARCA2 antibody

• Known target regions: Include primers for genomic regions known to be bound by SMARCA2

• Non-target regions: Regions not expected to have SMARCA2 binding

• Technical replicates: Minimum of three to assess reproducibility

For Immunofluorescence:

• Primary antibody omission: To assess secondary antibody background

• SMARCA2-depleted cells: Through siRNA or CRISPR approaches

• Peptide competition: Pre-incubation with immunizing peptide

• Co-localization controls: Nuclear markers to confirm expected localization

For Functional Studies:

• SMARCA2 knockdown controls: When studying antiviral effects, include appropriate controls such as those used in H5N1 virus experiments where SMARCA2 knockdown increased viral titer by >1 log₁₀

• Combined knockdowns: When studying synthetic lethality, include SMARCA2 knockdown alone, SMARCA4 knockdown alone, and combined knockdown

• Rescue experiments: Re-expression of SMARCA2 should rescue phenotypes if they are specifically due to SMARCA2 loss

How are SMARCA2 antibodies being used to develop and validate PROTAC-based therapeutic approaches?

SMARCA2 antibodies play crucial roles in the development and validation of Proteolysis Targeting Chimeras (PROTACs) as potential cancer therapeutics:

• Target engagement verification: Western blotting with SMARCA2 antibodies confirms whether PROTAC molecules like A947 successfully engage and degrade SMARCA2 protein. This quantitative assessment is essential for structure-activity relationship studies during PROTAC optimization .

• Selectivity assessment: Parallel Western blotting for SMARCA2 and SMARCA4 using specific antibodies is critical for demonstrating paralog selectivity. A947 demonstrated selective SMARCA2 degradation despite the high homology between these proteins, a finding that would be impossible to validate without selective antibodies .

• Temporal dynamics: Time-course studies using SMARCA2 antibodies reveal the kinetics of PROTAC-mediated degradation, informing dosing schedules for in vivo studies and potential clinical applications.

• In vivo confirmation: SMARCA2 antibodies are used in immunohistochemistry of tumor samples from PROTAC-treated animals to confirm target degradation in tissue contexts, correlating with efficacy measures .

• Mechanism elucidation: Immunoprecipitation with SMARCA2 antibodies followed by ubiquitin detection helps confirm the ubiquitin-proteasome pathway involvement in PROTAC-mediated degradation. Global ubiquitin mapping combined with proteome profiling revealed that A947 treatment did not cause unexpected off-target degradation .

This application demonstrates how SMARCA2 antibodies bridge fundamental chromatin biology research with targeted cancer therapeutic development.

What are the emerging roles of SMARCA2 in viral immunity and how can antibodies help investigate these functions?

Recent research has revealed unexpected roles for SMARCA2 in antiviral immunity, particularly against influenza viruses, opening new avenues for investigation:

• Indirect regulation of MxA activity: While SMARCA2 is not required for expression of the antiviral protein MxA itself, it is essential for MxA's antiviral activity against H5N1 and H7N7 influenza viruses. SMARCA2 antibodies in ChIP experiments can identify the direct transcriptional targets regulated by SMARCA2 that enable MxA function .

• Regulation of IFITM2 and IGFBP3: Transcriptome analysis identified these factors as SMARCA2-dependent genes required for efficient viral inhibition. Combining ChIP-Seq using SMARCA2 antibodies with RNA-Seq of SMARCA2-depleted cells can map the direct and indirect regulatory networks involved in this response .

• Cell-type specific functions: SMARCA2 antibodies enable investigation of tissue-specific roles in antiviral immunity through immunohistochemistry and cell sorting followed by ChIP-Seq, potentially revealing specialized functions in different immune cell populations.

• Chromatin dynamics during infection: Time-course ChIP-Seq with SMARCA2 antibodies during viral infection can reveal dynamic changes in chromatin occupancy, connecting chromatin remodeling events to transcriptional responses against pathogens.

• Therapeutic implications: Understanding SMARCA2's role in antiviral immunity using antibody-based approaches may reveal opportunities for enhancing immune responses or developing new antiviral strategies, particularly against influenza strains like H5N1 where SMARCA2 knockdown increased viral titer by >1 log₁₀ .

These emerging roles highlight the importance of chromatin regulation in host defense and demonstrate how SMARCA2 antibodies facilitate discovery at the intersection of epigenetics and immunology.

How can single-cell approaches utilizing SMARCA2 antibodies advance our understanding of heterogeneous cellular responses?

Single-cell technologies combined with SMARCA2 antibodies offer powerful new approaches to study cellular heterogeneity in development, disease, and therapeutic responses:

• Single-cell CUT&Tag/CUT&RUN: These techniques allow mapping of SMARCA2 chromatin occupancy in individual cells, revealing cell-to-cell variability in chromatin states that may underlie differential responses to stimuli or treatments. This is particularly relevant when studying heterogeneous systems like developing neural tissues, where SMARCA2 functions in both npBAF and nBAF complexes .

• Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq): Combining surface antibody labeling with single-cell RNA-seq allows correlation of SMARCA2 target gene expression with cell surface markers, enabling precise identification of cell populations with distinct SMARCA2 activity signatures.

• Single-cell multi-omics: Integrating SMARCA2 ChIP-seq with single-cell RNA-seq and ATAC-seq provides comprehensive views of how SMARCA2-mediated chromatin remodeling affects gene accessibility and expression at the individual cell level during processes like neural differentiation.

• Spatial transcriptomics with immunofluorescence: Combining SMARCA2 antibody staining with spatial transcriptomics techniques allows visualization of how SMARCA2 protein localization correlates with target gene expression in intact tissues, preserving spatial relationships between cells.

• Mass cytometry (CyTOF): Using metal-conjugated SMARCA2 antibodies in mass cytometry enables high-dimensional analysis of SMARCA2 levels alongside dozens of other proteins in individual cells, revealing how SMARCA2 expression correlates with cellular phenotypes and states.

These emerging single-cell approaches extend beyond population averages to reveal how SMARCA2-mediated chromatin remodeling contributes to cellular decision-making and phenotypic diversity, particularly in contexts like neural development and cancer heterogeneity.