SMARCA4 Monoclonal Antibody

What are the validated applications for SMARCA4/BRG1 antibodies?

SMARCA4/BRG1 antibodies have been extensively validated for multiple experimental approaches in molecular and cellular biology research. According to comprehensive testing, these antibodies perform reliably in Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence/Immunocytochemistry (IF/ICC), Immunoprecipitation (IP), Co-Immunoprecipitation (CoIP), Chromatin Immunoprecipitation (ChIP), RNA Immunoprecipitation (RIP), and ELISA applications . Published literature supports these applications with at least 25 publications using these antibodies for Western blotting, 9 for IHC, 7 for IF, and 7 for ChIP applications . This extensive validation across multiple techniques provides researchers with confidence in selecting the appropriate application for their specific experimental needs.

What are the recommended antibody dilutions for different experimental applications?

Optimal antibody dilution is critical for obtaining specific signals while minimizing background. Based on extensive validation, the following dilution ranges are recommended for SMARCA4/BRG1 antibodies:

These recommendations should be considered starting points, as optimal dilution may be sample-dependent. For instance, when working with the ZooMAb® mouse recombinant monoclonal antibody (clone 20C3.2), a higher dilution of 1:10,000 has proven effective for Western blotting in K562 cell lysates . It is advisable to perform titration experiments with your specific sample types to determine optimal conditions for maximum signal-to-noise ratio.

What is the species reactivity profile of commonly used SMARCA4 antibodies?

SMARCA4 antibodies demonstrate varying species reactivity profiles depending on the specific clone or product. The polyclonal antibody (21634-1-AP) has been extensively tested and confirmed to react with human, mouse, and rat samples . This cross-species reactivity is particularly valuable for comparative studies across model organisms. Citations in literature also suggest reactivity with zebrafish and bovine samples , expanding the potential research applications. Similarly, the ZooMAb® mouse recombinant monoclonal antibody (clone 20C3.2) and the Picoband® antibody also demonstrate reactivity with human, mouse, and rat samples . This consistent cross-species reactivity makes these antibodies versatile tools for researchers working with different model systems, facilitating translational research from animal models to human studies.

How should SMARCA4 antibodies be optimized for chromatin immunoprecipitation (ChIP) experiments?

ChIP experiments using SMARCA4 antibodies require careful optimization to ensure specific enrichment of SMARCA4-bound chromatin regions. Published research has successfully employed SMARCA4 antibodies in ChIP-seq experiments to identify direct regulatory targets, including the CCND1 (cyclin D1) promoter . When designing ChIP experiments with SMARCA4 antibodies, consider the following optimization strategies:

• Antibody amount: Use 2-5 μg of antibody per ChIP reaction for optimal chromatin pull-down efficiency.

• Chromatin fragmentation: Aim for fragments of 200-500 bp for high-resolution binding site identification.

• Controls: Include negative controls (IgG) and positive controls (known SMARCA4 binding sites such as the CCND1 promoter).

• Cross-validation: Verify ChIP-seq findings using ChIP-PCR for selected loci, as demonstrated in studies examining SMARCA4 occupancy at the CCND1 promoter in H1915 cells .

Research has shown that SMARCA4 binding at promoter regions is often associated with transcriptionally active genes marked by H3K27Ac, as evidenced by the correlation between SMARCA4 occupancy and H3K27Ac signal at the CCND1 promoter . This information can be valuable for interpreting ChIP results in the context of gene regulation mechanisms.

What strategies can resolve inconsistent SMARCA4 antibody staining in immunohistochemistry applications?

Inconsistent immunohistochemical staining with SMARCA4 antibodies can be attributed to several factors that require methodical optimization. Based on validated protocols, consider the following strategies:

• Antigen retrieval: SMARCA4 detection in FFPE tissues often requires heat-induced epitope retrieval. The recommended protocol involves TE buffer at pH 9.0, although citrate buffer at pH 6.0 may serve as an alternative for some tissue types . Comparative testing of both conditions may be necessary for your specific samples.

• Fixation variables: Overfixation can mask epitopes recognized by SMARCA4 antibodies. For prospective studies, standardize fixation time to 24 hours in 10% neutral buffered formalin.

• Antibody concentration: For challenging tissues, consider a starting dilution of 1:200 and perform a titration series (1:200, 1:400, 1:800) to identify optimal signal-to-noise ratio .

• Detection systems: Amplification systems like tyramide signal amplification may improve detection of low SMARCA4 expression, particularly in tumor samples with partial protein loss.

• Positive controls: Include known SMARCA4-positive tissues such as human colon cancer, lung cancer, or normal kidney tissues in each staining batch .

For research involving cancer specimens, be aware that SMARCA4 protein loss is a characteristic feature of certain tumors, particularly small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), with 54 of 61 SCCOHT tumors showing loss of SMARCA4 protein . Therefore, absence of staining in tumor cells with positive internal controls (e.g., endothelial cells) may represent a true biological finding rather than a technical failure.

How can researchers distinguish between true SMARCA4 protein loss and technical artifacts in cancer specimens?

Distinguishing between genuine SMARCA4 protein loss and technical artifacts is critical for accurate interpretation in cancer research. Implement this comprehensive validation approach:

• Internal controls: Evaluate SMARCA4 expression in non-neoplastic cells within the same tissue section, including endothelial cells and lymphocytes, which typically maintain SMARCA4 expression even when tumor cells show loss.

• Sequential sections: Perform SMARCA4 IHC on sequential sections to confirm consistent staining patterns and rule out tissue heterogeneity effects.

• Molecular correlation: Where possible, correlate IHC findings with SMARCA4 mutation status. Research shows that SMARCA4 protein loss strongly correlates with inactivating mutations, especially bi-allelic alterations . In a comprehensive study of SCCOHT tumors, 19 of 24 sequenced tumors harbored SMARCA4 mutations, and 16 of 19 stained tumors showed loss of SMARCA4 protein .

• Alternative antibody clones: Test multiple validated SMARCA4 antibody clones that recognize different epitopes. This approach helps confirm protein loss if multiple antibodies yield consistent negative results.

• Quantitative assessment: Use digital image analysis for objective quantification of nuclear SMARCA4 staining intensity, particularly in cases with partial loss or reduced expression levels.

It's important to note that some SMARCA4-negative tumors may carry heterozygous mutations or lack identifiable sequence alterations in the SMARCA4 gene , suggesting that mechanisms beyond coding mutations may lead to protein loss, including epigenetic silencing or post-transcriptional regulation.

How can SMARCA4 antibodies be used to investigate synthetic lethality in cancer models?

SMARCA4 antibodies serve as critical tools for characterizing synthetic lethal interactions in cancer research, particularly in identifying therapeutic vulnerabilities in SMARCA4-deficient tumors. To implement this research approach:

• Verification of SMARCA4 status: First establish SMARCA4 expression status in cell line models using Western blotting with validated antibodies at dilutions of 1:500-1:3000 . This baseline characterization is essential for subsequent synthetic lethality experiments.

• Functional validation: In SMARCA4-deficient models, confirm absence of chromatin remodeling activity at SMARCA4-dependent loci using ChIP assays targeting known SMARCA4-regulated genes such as CCND1 .

• Drug sensitivity correlation: Research has demonstrated that SMARCA4 deficiency correlates with increased sensitivity to CDK4/6 inhibitors such as palbociclib in non-small cell lung cancer (NSCLC) models . When testing drug responses, include both short-term viability assays and long-term colony formation assays to comprehensively assess synthetic lethal effects.

• Mechanistic investigation: SMARCA4 deficiency has been linked to reduced cyclin D1 expression, which contributes to CDK4/6 inhibitor sensitivity . Monitor cyclin D1 levels using appropriate antibodies in parallel with SMARCA4 detection to establish this mechanistic link in your experimental system.

• Rescue experiments: To confirm specificity of synthetic lethal interactions, perform rescue experiments by re-expressing SMARCA4 in deficient cells. For example, ectopic cyclin D1 expression has been shown to confer palbociclib resistance in SMARCA4-deficient H1299 and SMARCA4/2-dual deficient H1703 cells .

This research approach has significant translational implications, as it identifies CDK4/6 inhibitors as potential therapeutic agents for SMARCA4-deficient cancers, provided RB expression remains intact .

What are the technical considerations when using SMARCA4 antibodies for cancer diagnostic applications?

While research applications of SMARCA4 antibodies are well-established, their potential diagnostic utility requires rigorous validation and standardization. Consider these technical aspects:

The diagnostic utility of SMARCA4 antibodies continues to evolve as additional research clarifies the relationship between SMARCA4 status and therapeutic response in various cancer types.

What quality control measures are essential when validating SMARCA4 antibody specificity?

Rigorous quality control is critical for ensuring reliable results with SMARCA4 antibodies. Implement these comprehensive validation measures:

• Positive and negative controls: Include cell lines with known SMARCA4 expression status. HeLa, HepG2, MCF-7, and PC-3 cells have been validated as positive controls for SMARCA4 detection . For negative controls, use SMARCA4-deficient cell lines such as those identified in NSCLC (e.g., H1299) or SCCOHT (e.g., BIN-67) .

• Knockdown/knockout validation: Confirm antibody specificity using SMARCA4 knockdown or knockout models. Literature reports have validated SMARCA4 antibodies using such approaches, with at least 4 publications demonstrating specificity through KD/KO experiments .

• Molecular weight confirmation: In Western blotting applications, verify detection of the expected 185 kDa band corresponding to SMARCA4/BRG1 . Observed molecular weight should align with the calculated molecular weight of 185 kDa for the 1647 amino acid protein.

• Cross-reactivity assessment: Test for potential cross-reactivity with related SWI/SNF complex components, particularly SMARCA2/BRM, which shares structural similarities with SMARCA4/BRG1.

• Multiple application validation: Confirm consistent results across different applications (e.g., WB, IHC, IF) using the same antibody. Commercial SMARCA4 antibodies have been validated across multiple applications, providing stronger evidence for specificity .

• Lot-to-lot consistency: When using commercial antibodies, test new lots against previously validated lots to ensure consistent performance. This is particularly important for polyclonal antibodies, which may show greater lot-to-lot variation compared to monoclonal antibodies.

Implementation of these quality control measures helps ensure that experimental observations truly reflect SMARCA4 biology rather than artifacts of antibody cross-reactivity or non-specific binding.

How should researchers optimize storage and handling of SMARCA4 antibodies to maintain activity?

Proper storage and handling are crucial for maintaining antibody performance over time. Based on manufacturer recommendations and best practices, follow these guidelines:

• Storage temperature: Store SMARCA4 antibodies at -20°C for long-term storage (up to one year from receipt). After reconstitution, store at 4°C for short-term use (up to one month) . Lyophilized antibodies generally have greater stability than those in liquid formulation.

• Buffer composition: SMARCA4 antibodies are typically stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . This formulation helps maintain antibody stability during freeze-thaw cycles.

• Aliquoting: For antibodies requiring multiple freeze-thaw cycles, prepare small aliquots upon initial thawing to avoid repeated freezing and thawing of the entire stock. For some formulations, aliquoting may be unnecessary for -20°C storage, particularly for smaller volumes (20 μl) containing 0.1% BSA .

• Freeze-thaw cycles: Minimize freeze-thaw cycles as they can lead to antibody denaturation and reduced activity. According to manufacturer guidelines, repeated freeze-thaw cycles should be avoided .

• Working dilutions: Prepare working dilutions immediately before use and do not store diluted antibodies for extended periods, as protein adsorption to tube walls and microbial contamination can reduce effectiveness.

• Handling precautions: Sodium azide, a common preservative in antibody solutions, is toxic and can react with lead and copper plumbing to form explosive metal azides. When disposing of such solutions, flush with large volumes of water to prevent azide accumulation.

By following these storage and handling recommendations, researchers can maximize the lifespan and consistent performance of SMARCA4 antibodies, ensuring reliable experimental results over time.

How can SMARCA4 antibodies be leveraged in epigenetic research and chromatin dynamics studies?

SMARCA4 antibodies are increasingly valuable tools for investigating epigenetic mechanisms and chromatin dynamics, with several advanced applications:

• Sequential ChIP (Re-ChIP): For identifying genomic loci where SMARCA4 co-localizes with other transcription factors or chromatin modifiers, sequential ChIP experiments can be performed. This approach has helped elucidate the cooperative interaction between SMARCA4 and transcription factors at regulatory regions.

• CUT&RUN and CUT&Tag: These newer techniques offer advantages over traditional ChIP by providing higher signal-to-noise ratios and requiring fewer cells. SMARCA4 antibodies compatible with these methods allow mapping of SMARCA4 binding with greater precision and from limited biological samples.

• Chromatin accessibility correlation: Combining SMARCA4 ChIP-seq with ATAC-seq has revealed relationships between SMARCA4 binding and chromatin accessibility. Research has documented ATAC-seq signal in the CCND1 locus in relation to SMARCA4 occupancy, demonstrating the functional impact of SMARCA4 on chromatin structure .

• Live-cell imaging: Emerging applications involve using SMARCA4 antibody fragments or nanobodies for tracking SMARCA4 dynamics in living cells, providing temporal information about SWI/SNF complex assembly and function.

• Single-cell approaches: Adapting SMARCA4 antibodies for single-cell technologies such as CUT&Tag-seq or Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) enables investigation of heterogeneity in SMARCA4 function across individual cells within a population.

These emerging applications expand the utility of SMARCA4 antibodies beyond traditional protein detection methods, enabling more sophisticated investigations of chromatin biology and gene regulation mechanisms.

What is the current understanding of SMARCA4 mutations in cancer, and how can antibodies help characterize these deficiencies?

SMARCA4 mutations occur in approximately 4% of all cancers, with notable frequency in specific tumor types. SMARCA4 antibodies are instrumental in characterizing the functional consequences of these mutations:

Understanding the landscape of SMARCA4 mutations across cancer types continues to evolve, with implications for both diagnosis and targeted therapeutic approaches.