SMARCE1 Antibody, Biotin conjugated

What is SMARCE1 and why is it significant in chromatin biology research?

SMARCE1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily E member 1) functions as a key component of SWI/SNF chromatin remodeling complexes that carry out enzymatic activities to alter chromatin structure by changing DNA-histone contacts within nucleosomes in an ATP-dependent manner . The protein plays dual roles in both transcriptional activation and repression of select genes through chromatin remodeling, altering DNA-nucleosome topology . SMARCE1 has particular significance in neural development, as it belongs to both neural progenitor-specific chromatin remodeling complexes (npBAF) and neuron-specific chromatin remodeling complexes (nBAF) . During neural development, as progenitor cells exit mitosis and differentiate into neurons, the composition of these BAF complexes changes, with SMARCE1 participating in complexes that are essential for neural stem cell self-renewal and dendrite growth regulation .

What experimental applications is the biotin-conjugated SMARCE1 antibody suitable for?

The biotin-conjugated SMARCE1 recombinant antibody is validated for multiple experimental applications, providing versatility in research settings. According to technical specifications, the antibody is suitable for Western Blotting (WB) at dilutions of 1:300-5000, Immunohistochemistry on paraffin-embedded tissues (IHC-P) at 1:200-400, Immunohistochemistry on frozen tissues (IHC-F) at 1:100-500, and Immunoprecipitation (IP) using 1-2μg of antibody . While not directly listed for the biotin-conjugated version, related SMARCE1 antibodies have also demonstrated efficacy in Chromatin Immunoprecipitation (ChIP) and Immunocytochemistry/Immunofluorescence (ICC/IF) applications . The biotin conjugation provides additional versatility through compatibility with streptavidin-based detection systems, enhancing sensitivity in signal amplification scenarios.

How should researchers prepare samples for optimal SMARCE1 detection?

For optimal SMARCE1 detection, sample preparation protocols should be tailored to the specific application and cellular localization of the protein. Since SMARCE1 functions primarily in the nucleus as part of chromatin remodeling complexes, nuclear extraction protocols are recommended for applications like Western blotting and immunoprecipitation . For tissue sections in IHC-P applications, appropriate antigen retrieval methods are crucial - heat-induced epitope retrieval in citrate buffer (pH 6.0) is typically effective for exposing the SMARCE1 epitope . When performing immunofluorescence, fixation with 4% paraformaldehyde followed by permeabilization with 0.1-0.5% Triton X-100 generally preserves SMARCE1 antigenicity while allowing antibody access to the nuclear compartment . For ChIP applications, crosslinking with 1% formaldehyde for 10 minutes at room temperature followed by sonication to generate DNA fragments of 200-500bp has been successfully employed with similar SMARCE1 antibodies .

How can the SMARCE1 antibody be utilized in cancer diagnostic research?

SMARCE1 immunohistochemistry has emerged as a highly specific diagnostic biomarker for clear cell meningioma (CCM), a rare grade II histopathological subtype associated with high recurrence rates. Multiple studies have demonstrated that loss of SMARCE1 immunoexpression is highly specific to CCM, with preservation of nuclear immunostaining in all other meningioma variants and non-meningioma clear cell tumors . This diagnostic application is particularly valuable because traditional histopathological distinction between CCM and other meningioma subtypes can be challenging. In validation studies, SMARCE1 immunostaining showed perfect correlation with molecular analysis of SMARCE1 gene status, with all CCMs demonstrating loss of SMARCE1 expression corresponding to bi-allelic inactivating events detected by NGS-based sequencing . The biotin-conjugated format of the antibody may offer enhanced sensitivity in this diagnostic application through signal amplification with streptavidin detection systems, particularly in cases with limited tissue availability.

What methodological approaches can researchers use to study SMARCE1's role in metastasis?

SMARCE1 has been implicated in regulating the metastatic potential of breast cancer cells, making it an important target for understanding cancer progression mechanisms . To investigate SMARCE1's role in metastasis, researchers can employ several methodological approaches using the biotin-conjugated antibody:

• Expression correlation studies: Immunohistochemistry can be used to analyze SMARCE1 expression levels across primary tumors and matched metastatic lesions, correlating expression patterns with clinical outcomes .

• Functional genomics approach: Combining SMARCE1 knockdown or overexpression with metastasis assays, including:

  • In vitro migration and invasion assays

  • Anoikis resistance testing in nonadherent culture conditions

  • In vivo metastasis models using xenografts with subsequent antibody-based detection of metastatic foci

• Mechanistic studies: ChIP experiments utilizing SMARCE1 antibodies can identify genomic targets directly regulated by SMARCE1-containing complexes in metastatic versus non-metastatic cells .

• Protein interaction networks: Immunoprecipitation with SMARCE1 antibodies followed by mass spectrometry can identify differential protein interactions in metastatic contexts, particularly focusing on interactions with the focal adhesion kinase (FAK/PTK2) pathway, which has been implicated as a downstream effector of SMARCE1 in metastasis regulation .

How can researchers optimize ChIP protocols specifically for SMARCE1?

Chromatin immunoprecipitation (ChIP) using SMARCE1 antibodies presents unique challenges due to SMARCE1's function within large chromatin remodeling complexes. An optimized protocol should include:

• Crosslinking optimization: Standard 1% formaldehyde crosslinking for 10 minutes may be insufficient to capture interactions within large protein complexes like SWI/SNF. Dual crosslinking with 1mM disuccinimidyl glutarate (DSG) for 30 minutes followed by 1% formaldehyde for 10 minutes can improve capture of SMARCE1-containing complexes .

• Sonication parameters: Due to SMARCE1's association with regions of altered chromatin structure, standard sonication protocols may require adjustment. Aim for fragments between 200-500bp, but assess sonication efficiency through pilot experiments comparing different durations and power settings .

• Antibody concentration: For biotin-conjugated SMARCE1 antibody, higher concentrations than used for IP may be necessary (typically 3-5μg per ChIP reaction) due to the complex chromatin environment. Pre-clearing with protein A/G beads and non-relevant IgG is crucial to reduce background .

• Washing stringency: Due to the biotin conjugation, washing stringency must be carefully optimized to prevent loss of specific signal while reducing background. A gradual increase in salt concentration during wash steps (from 150mM to 500mM NaCl) can help achieve this balance .

• Detection system: When using biotin-conjugated antibodies for ChIP, researchers must avoid streptavidin-based detection systems for pulldown to prevent interference from the biotin conjugation. Instead, protein A/G beads coupled with anti-rabbit secondary antibodies can be used for immunoprecipitation .

What is known about SMARCE1's role in neural development and how can the antibody be used to investigate it?

SMARCE1 plays distinct roles in neural progenitors and differentiated neurons through its participation in stage-specific BAF complexes (npBAF and nBAF). During neural development, a critical switch from stem/progenitor chromatin remodeling mechanisms to postmitotic neuronal mechanisms occurs as neurons exit the cell cycle . This transition involves changes in BAF complex composition, with SMARCE1 participating in both complexes but interacting with different partners.

Researchers can utilize biotin-conjugated SMARCE1 antibodies to investigate these developmental roles through:

• Developmental expression profiling: IHC or IF studies tracking SMARCE1 localization and expression levels across neural development stages, comparing with markers of neural progenitors versus differentiated neurons .

• Co-immunoprecipitation studies: Using the antibody to pull down SMARCE1-containing complexes at different developmental stages to identify stage-specific interaction partners through mass spectrometry or Western blotting for known npBAF components (ACTL6A/BAF53A, PHF10/BAF45A) versus nBAF components (ACTL6B/BAF53B, DPF1/BAF45B, DPF3/BAF45C) .

• ChIP-seq experiments: Mapping SMARCE1 genomic binding sites in neural progenitors versus differentiated neurons to identify differential regulatory targets and correlate with gene expression changes during differentiation .

• Functional studies: Combining SMARCE1 knockdown or overexpression with differentiation assays, using the antibody to verify expression changes and correlate with phenotypic outcomes in dendrite growth and neuronal maturation .

What controls should be included when using biotin-conjugated SMARCE1 antibody?

A robust experimental design using biotin-conjugated SMARCE1 antibody should incorporate multiple controls to ensure specificity and validity of results:

For diagnostic applications in clear cell meningioma, include known positive controls (other meningioma subtypes showing nuclear staining) and known negative controls (confirmed CCM cases) .

How can researchers address background issues when using biotin-conjugated antibodies?

Biotin-conjugated antibodies can present unique background challenges due to endogenous biotin in biological samples and the high affinity of the biotin-streptavidin interaction. To minimize background:

• Endogenous biotin blocking: Prior to primary antibody incubation, block endogenous biotin using commercially available biotin-blocking kits, which typically involve sequential incubation with avidin and biotin .

• Sample preparation optimization: For tissues rich in endogenous biotin (liver, kidney, brain), consider alternative fixation methods or increase the concentration of blocking reagents. For IHC-P applications, avoid biotin-based antigen retrieval systems .

• Dilution optimization: While the recommended dilution range for IHC applications is 1:200-400, systematic titration experiments should be performed for each new application or tissue type to determine optimal signal-to-noise ratio .

• Alternative detection systems: In particularly challenging samples, consider indirect detection methods where a non-conjugated primary SMARCE1 antibody is followed by a biotinylated secondary antibody, allowing for more stringent washing conditions .

• Washing optimization: Extend washing steps after primary antibody incubation using buffers containing 0.1-0.3% Tween-20 to reduce non-specific binding without disrupting specific interactions .

How do researchers differentiate between specific and non-specific signals in SMARCE1 immunostaining?

Distinguishing specific from non-specific signals is critical for accurate interpretation of SMARCE1 immunostaining results:

• Knowledge of expected localization: SMARCE1 should display predominantly nuclear localization as a component of chromatin remodeling complexes. Cytoplasmic staining generally indicates non-specific binding .

• Pattern recognition: Specific SMARCE1 staining typically shows homogeneous or slightly granular nuclear distribution, while non-specific staining often presents as irregular, intense spots or diffuse cytoplasmic staining .

• Context-dependent interpretation: In clear cell meningioma diagnosis, complete loss of nuclear SMARCE1 expression is the diagnostic pattern, contrasting with preserved nuclear staining in other meningioma subtypes. This binary pattern aids in distinguishing specific from non-specific signals .

• Quantitative assessment: For research applications, quantifying nuclear to cytoplasmic signal ratio can help establish thresholds for specific signal determination. Automated image analysis using nuclear counterstains as reference can facilitate this process .

• Complementary techniques: Validate unexpected immunostaining patterns with orthogonal techniques like Western blotting or qRT-PCR to confirm expression patterns observed in immunohistochemistry .

How can SMARCE1 antibody staining be standardized for potential diagnostic applications?

Given the emerging role of SMARCE1 as a diagnostic biomarker for clear cell meningioma, standardization of immunostaining protocols is essential for potential clinical implementation:

• Antigen retrieval standardization: Heat-induced epitope retrieval using citrate buffer (pH 6.0) has been validated in multiple studies. Standardize heating times (20 minutes) and cooling periods (20 minutes at room temperature) to ensure consistent epitope exposure .

• Scoring system development: Implement a semi-quantitative scoring system:

  • Positive: Strong nuclear staining in >90% of tumor cells

  • Reduced: Weak or patchy nuclear staining in 10-90% of tumor cells

  • Negative: Complete absence or staining in <10% of tumor cells

• Internal controls: Utilize non-neoplastic cells within the specimen (endothelial cells, inflammatory cells) as internal positive controls for SMARCE1 expression .

• Reference standards: Include reference tissue microarrays with known SMARCE1 expression patterns to calibrate batch-to-batch staining intensity .

• Automated detection: Develop digital pathology algorithms for standardized quantification of SMARCE1 nuclear staining intensity and distribution, reducing inter-observer variability .

• Correlation with molecular testing: Establish concordance rates between immunohistochemical SMARCE1 loss and molecular detection of SMARCE1 mutations or deletions through NGS-based sequencing .

What are the latest findings regarding SMARCE1's role in cancer progression beyond meningioma?

Recent research has expanded understanding of SMARCE1's role in multiple cancer types:

• Breast cancer metastasis: SMARCE1 has been identified as a regulator of metastatic potential in breast cancer cells. Mechanistic studies have linked SMARCE1 to the regulation of genes involved in cell adhesion and migration, particularly through modulation of the focal adhesion kinase (FAK/PTK2) pathway . Loss of SMARCE1 function reduced migratory capacity and anchorage-independent growth of breast cancer cells, suggesting context-dependent roles in cancer progression .

• Hormone-responsive cancers: SMARCE1 is required for coactivation of estrogen-responsive promoters by SWI/SNF complexes and the SRC/p160 family of histone acetyltransferases, suggesting a specific role in hormone-dependent cancers . This function provides a mechanistic link between chromatin remodeling and hormone receptor signaling networks frequently dysregulated in cancer.

• Neural reprogramming: SMARCE1 interacts with the CoREST corepressor, resulting in repression of neuron-specific gene promoters in non-neuronal cells . Dysregulation of this mechanism may contribute to aberrant cellular differentiation states observed in aggressive cancers.

• Therapeutic implications: The specificity of SMARCE1 loss in clear cell meningioma suggests potential for targeted therapeutic approaches. Early-stage investigations are exploring synthetic lethality approaches targeting residual SWI/SNF complex dependencies in SMARCE1-deficient tumors .

What is the most effective protein extraction method for detecting SMARCE1 in Western blot applications?

Effective detection of SMARCE1 in Western blot applications requires optimization of protein extraction to preserve nuclear proteins while minimizing proteolytic degradation:

• Nuclear extraction protocol:

  • Lyse cells in hypotonic buffer (10mM HEPES pH 7.9, 10mM KCl, 1.5mM MgCl₂) with protease inhibitors

  • Disrupt cell membranes with 0.5% NP-40

  • Isolate nuclei by centrifugation (3000g, 10 minutes)

  • Extract nuclear proteins with high-salt buffer (20mM HEPES pH 7.9, 420mM NaCl, 1.5mM MgCl₂, 0.2mM EDTA, 25% glycerol)

• Denaturing conditions: SMARCE1 is part of large protein complexes; complete denaturation requires 2% SDS and boiling at 95°C for 5 minutes to ensure detection of the monomeric form (predicted molecular weight ~46-47kDa) .

• Sample handling: SMARCE1 is susceptible to proteolytic degradation. Process samples at 4°C, include protease inhibitor cocktail, and avoid repeated freeze-thaw cycles .

• Loading controls: Traditional housekeeping proteins may not be appropriate when comparing nuclear extracts. Consider nuclear-specific loading controls like Lamin B1 or histone H3 .

• Gel percentage: Use 10-12% polyacrylamide gels for optimal resolution of SMARCE1 protein, which typically appears as a band at approximately 46-47 kDa .

How does biotin conjugation affect antibody performance compared to unconjugated alternatives?

Biotin conjugation introduces specific considerations that differentiate the performance of biotin-conjugated SMARCE1 antibodies from their unconjugated counterparts:

For applications requiring maximum sensitivity (low abundance targets) or multiplexing capabilities, the biotin-conjugated format often provides advantages that outweigh the additional controls required .

How might SMARCE1 antibodies contribute to emerging therapeutic approaches targeting chromatin remodeling?

As understanding of chromatin remodeling in disease progression advances, SMARCE1 antibodies will likely play important roles in developing and validating new therapeutic approaches:

• Target validation: SMARCE1 antibodies can help validate the specificity of small molecule inhibitors being developed to target SWI/SNF complex components by assessing binding effects on complex composition and stability .

• Biomarker development: Given the specificity of SMARCE1 loss in clear cell meningioma, SMARCE1 immunostaining may serve as a companion diagnostic for future targeted therapies, helping stratify patients for clinical trials .

• Therapeutic resistance mechanisms: Chromatin remodeling complexes have been implicated in resistance to various cancer therapies. SMARCE1 antibodies can help map changes in SWI/SNF complex composition during treatment and relapse .

• Epigenetic reprogramming approaches: As therapies targeting epigenetic mechanisms advance, SMARCE1 antibodies will be valuable tools for assessing how these interventions affect SWI/SNF complex recruitment and activity at target genomic loci .

• Cell-free chromatin studies: Emerging liquid biopsy approaches analyzing cell-free chromatin may incorporate SMARCE1 antibodies to assess chromatin accessibility patterns that reflect disease states or treatment responses .