SMARCE1 Antibody, HRP conjugated

What is SMARCE1 and why is it important in research?

SMARCE1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily E member 1) is a component of the ATP-dependent chromatin remodeling complex SWI/SNF. This complex is required for transcriptional activation of genes normally inhibited by chromatin. SMARCE1 plays a critical role in the invasive progression of early-stage cancers, including ductal carcinoma in situ (DCIS), by regulating the expression of secreted proteases that degrade basement membrane . Research has shown that SMARCE1 forms a SWI/SNF-independent complex with the transcription factor ILF3 specifically in invasive cells, pointing to its mechanistic role in cancer progression . SMARCE1 is particularly important in research due to its strong correlation with relapse and metastasis in patients diagnosed with early-stage cancers .

What are the common applications for SMARCE1 antibodies in research?

SMARCE1 antibodies are utilized in multiple experimental applications including:

These applications allow researchers to investigate SMARCE1's expression, localization, and interactions in various experimental contexts, particularly in cancer research .

How does an HRP-conjugated SMARCE1 antibody differ from unconjugated versions?

HRP-conjugated SMARCE1 antibodies have horseradish peroxidase directly attached to the antibody molecule, whereas unconjugated versions (as described in the search results) require a secondary detection system . The primary advantages of HRP-conjugated antibodies include:

• Streamlined experimental workflow by eliminating the secondary antibody incubation step

• Reduced background signal due to fewer cross-reactivity issues

• Potentially higher sensitivity in certain applications

• Direct visualization through enzymatic conversion of substrates

When selecting between conjugated and unconjugated antibodies, researchers should consider that unconjugated antibodies offer greater flexibility with detection systems and signal amplification strategies, while HRP-conjugated versions provide workflow efficiency and potentially cleaner results in certain experimental contexts.

What are the optimal dilutions for HRP-conjugated SMARCE1 antibodies in different applications?

While specific dilutions for HRP-conjugated SMARCE1 antibodies are not provided in the search results, we can extrapolate from the unconjugated antibody recommendations with adjustments typical for conjugated antibodies:

Important methodological considerations include:

• Always optimize dilutions for each specific application and sample type

• HRP-conjugated antibodies typically require higher dilutions than unconjugated versions due to direct detection

• Sample-dependent variations may necessitate empirical determination of optimal conditions

• Antigen retrieval methods significantly impact results in IHC applications (TE buffer pH 9.0 or citrate buffer pH 6.0 are recommended)

What controls should be included when using SMARCE1 antibodies in cancer research?

For rigorous experimental design with SMARCE1 antibodies, researchers should include:

• Positive Controls:

  • Cell lines with confirmed SMARCE1 expression: Jurkat, HEK-293, HeLa, and MCF-7 cells

  • Tissue samples: Human prostate cancer or breast cancer tissues with known SMARCE1 expression

• Negative Controls:

  • Primary antibody omission control

  • Isotype control (Mouse IgG1 for monoclonal or Rabbit IgG for polyclonal antibodies)

  • SMARCE1 knockdown samples (if available)

• Technical Controls:

  • Loading controls for Western blot (e.g., GAPDH, β-actin)

  • Tissue controls with varying SMARCE1 expression levels

  • Peptide competition assays to confirm specificity

These controls are essential for validating experimental findings, particularly when investigating SMARCE1's role in tumor invasion and metastasis, as described in the research demonstrating SMARCE1's function in early-stage cancer progression .

How should sample preparation be optimized for SMARCE1 detection in different tissue types?

Sample preparation protocols should be tailored to specific tissue types and applications:

For Western Blot Analysis:

• Use RIPA or NP-40 based lysis buffers with protease inhibitors

• Ensure complete tissue homogenization or cell lysis

• Include phosphatase inhibitors if phosphorylation status is relevant

• Load 20-50 μg of total protein per lane

• Expected molecular weight: 50-55 kDa (observed) vs. 47 kDa (calculated)

For Immunohistochemistry:

• Formalin-fixed paraffin-embedded (FFPE) tissue sections (4-6 μm)

• Critical Step: Perform antigen retrieval with TE buffer pH 9.0 as recommended; alternatively, citrate buffer pH 6.0 may be used

• Blocking with appropriate serum (5-10%) to reduce background

• Primary antibody incubation at optimal dilution (1:20-1:200 for unconjugated versions; adjust accordingly for HRP-conjugated)

• For tissue microarrays containing breast cancer samples, consider the progressive increase in SMARCE1 expression during tumor progression

For Immunofluorescence:

• Fix cells with 4% paraformaldehyde

• Permeabilize with 0.1-0.5% Triton X-100

• Block with 1-5% BSA or normal serum

• Use appropriate dilution (1:200-1:800 for unconjugated versions; adjust accordingly for HRP-conjugated)

• Include nuclear counterstain (e.g., DAPI) to assess nuclear localization

What are common causes of false negative or weak signals when using SMARCE1 antibodies?

When encountering weak or absent signals with SMARCE1 antibodies, consider these potential issues and solutions:

When investigating SMARCE1 in tumor invasion models, consider that expression levels may vary significantly between invasive and non-invasive cell populations, as demonstrated by studies showing SMARCE1's specific role in invasive progression .

How can researchers validate the specificity of SMARCE1 antibody signals?

Rigorous validation strategies include:

• Molecular Approaches:

  • siRNA or shRNA knockdown of SMARCE1 (as used in studies examining SMARCE1's role in invasion)

  • CRISPR-Cas9 knockout controls

  • Overexpression systems with tagged SMARCE1

• Antibody-Specific Methods:

  • Peptide competition assays using the immunogen (SMARCE1 fusion protein Ag1161)

  • Comparison of staining patterns with multiple antibodies targeting different SMARCE1 epitopes

  • Correlation of results across multiple detection methods (e.g., WB, IHC, IF)

• Application-Specific Validations:

  • For IHC: Comparison of staining in tissues with known SMARCE1 expression patterns

  • For WB: Confirmation of the expected 50-55 kDa band size

  • For IP: Validation of pulled-down proteins by mass spectrometry, as demonstrated in studies identifying SMARCE1-ILF3 interactions

How should researchers interpret varying SMARCE1 expression levels across different cancer stages?

Research has demonstrated that SMARCE1 expression increases during tumor progression and correlates with invasive potential . When interpreting SMARCE1 expression data:

• Progression Analysis:

  • SMARCE1 expression is lowest in early-stage breast cancers

  • Expression increases during tumor progression

  • Highest levels observed in tumors invading adjacent lymph nodes

• Prognostic Implications:

  • High SMARCE1 expression in early-stage tumors may indicate increased metastatic potential

  • Consider correlating SMARCE1 expression with clinical outcomes and invasion markers

  • SMARCE1 can serve as a biomarker to prospectively identify tumors with metastatic propensity

• Mechanistic Considerations:

  • Evaluate SMARCE1 in context of basement membrane degradation capacity

  • Consider co-expression with matrix metalloproteinases (MMPs)

  • Assess association with ILF3, as SMARCE1 forms a SWI/SNF-independent complex with ILF3 specifically in invasive cells

How can SMARCE1 antibodies be utilized to investigate chromatin remodeling complexes?

SMARCE1 antibodies are powerful tools for dissecting the composition and function of SWI/SNF complexes:

• Protein Complex Analysis:

  • Co-immunoprecipitation (Co-IP) to isolate SMARCE1-containing complexes

  • Proximity ligation assays to visualize SMARCE1 interactions with other SWI/SNF components in situ

  • ChIP-seq to map SMARCE1 binding sites across the genome

• Functional Studies:

  • ChIP-qPCR to quantify SMARCE1 enrichment at specific promoters

  • Sequential ChIP (Re-ChIP) to identify genomic regions bound by specific SMARCE1-containing complexes

  • ATAC-seq or DNase-seq following SMARCE1 manipulation to assess chromatin accessibility changes

• Differential Complex Analysis:

  • IP-MS approaches to distinguish between canonical SWI/SNF complexes and alternative SMARCE1-containing complexes

  • Compare SMARCE1 and SMARCC1 immunoprecipitates to identify SWI/SNF-independent SMARCE1 partners, similar to the approach that identified the SMARCE1-ILF3 interaction in invasive cells

What methodologies can be employed to study SMARCE1's role in cancer invasion and metastasis?

Based on established research methodologies , several approaches can effectively investigate SMARCE1's function in invasion:

• 3D Invasion Models:

  • Basement membrane culture systems to assess spheroid invasion

  • Quantification of invasion stages (non-invasive, partially invasive, highly invasive)

  • Integration of fluorescent collagen IV substrates to measure matrix metalloproteinase activity during invasion

• In Vivo Metastasis Assays:

  • Orthotopic mouse models with SMARCE1-modulated cells

  • Quantification of circulating tumor cells following SMARCE1 inhibition

  • Assessment of local tumor invasion and distant metastasis

  • Tail-vein injection models to study later stages of the metastatic cascade

• Molecular Mechanism Studies:

  • IP-MS to identify SMARCE1 binding partners in invasive versus non-invasive contexts

  • RNA-seq to define SMARCE1-dependent transcriptional programs

  • Protease activity assays to measure basement membrane degradation capacity

  • Investigation of SMARCE1-ILF3 complex formation and its transcriptional targets

How can researchers distinguish between SWI/SNF-dependent and independent functions of SMARCE1?

Distinguishing between SMARCE1's canonical SWI/SNF-related functions and its SWI/SNF-independent activities requires sophisticated approaches:

• Comparative Proteomics:

  • Parallel IP-MS of multiple SWI/SNF components (e.g., SMARCE1 and SMARCC1)

  • Identification of proteins uniquely bound to SMARCE1 but not core SWI/SNF components

  • Temporal analysis of complex formation during biological processes like invasion

• Targeted Disruption Strategies:

  • Mutation of specific SMARCE1 domains to selectively disrupt either SWI/SNF incorporation or alternative interactions

  • siRNA knockdown of core SWI/SNF components with SMARCE1 overexpression to isolate SWI/SNF-independent functions

  • CRISPR-based approaches to modify endogenous SMARCE1 interaction domains

• Functional Genomics:

  • ChIP-seq comparison of SMARCE1 and core SWI/SNF components to identify unique SMARCE1 binding sites

  • Transcriptome analysis following selective disruption of SWI/SNF-dependent versus independent interactions

  • Assessment of ILF3 binding motifs in SMARCE1-regulated genes, as the SMARCE1-ILF3 complex represents a documented SWI/SNF-independent function

What detection systems work optimally with HRP-conjugated SMARCE1 antibodies?

When using HRP-conjugated SMARCE1 antibodies, selection of an appropriate detection system is critical:

• Western Blot Detection:

  • Enhanced chemiluminescence (ECL) substrates offer good sensitivity

  • Extended duration ECL substrates for weak signals

  • Quantitative fluorescent substrates for precise quantification

• IHC Detection Options:

  • DAB (3,3'-diaminobenzidine) produces a brown precipitate suitable for brightfield microscopy

  • AEC (3-amino-9-ethylcarbazole) yields a red precipitate that's alcohol-soluble

  • Tyramide signal amplification (TSA) systems for enhanced sensitivity in detecting low-abundance SMARCE1

• Methodological Considerations:

  • Substrate incubation times should be optimized for each experiment

  • Signal-to-noise ratio can be improved by titrating antibody concentration

  • For multiplexing, consider spectral unmixing approaches or sequential detection protocols

What are best practices for storage and handling of HRP-conjugated antibodies to maintain activity?

Proper storage and handling are essential for maintaining the activity of HRP-conjugated antibodies:

• Storage Recommendations:

  • Store at -20°C in small aliquots to prevent freeze-thaw cycles

  • For working solutions, store at 4°C for up to 1 month

  • Include carrier proteins (0.1% BSA) for dilute antibody solutions

  • Avoid storage buffers containing sodium azide, which inhibits HRP activity

• Handling Guidelines:

  • Minimize exposure to light and oxidizing agents

  • Avoid repeated freeze-thaw cycles (more than 3-5 cycles significantly reduces activity)

  • Centrifuge briefly before opening vials to collect solution at the bottom

  • Use sterile technique when handling antibody solutions

• Quality Control:

  • Periodically test activity using positive control samples

  • Monitor for signs of degradation (decreased signal intensity, increased background)

  • Document lot numbers and performance metrics for reproducibility

How should researchers quantify and normalize SMARCE1 expression in comparative studies?

For robust quantitative analysis of SMARCE1 expression:

• Western Blot Quantification:

  • Use validated housekeeping proteins (β-actin, GAPDH) as loading controls

  • Employ digital image analysis software for densitometry

  • Establish linear range of detection for accurate quantification

  • Present data as relative expression normalized to controls

• IHC Quantification Approaches:

  • H-score method (intensity × percentage positive cells)

  • Digital image analysis with positive pixel algorithms

  • Consider nuclear versus cytoplasmic localization separately

  • Compare with established scoring systems used in cancer progression studies

• Multi-method Validation:

  • Correlate protein expression with mRNA levels

  • Validate findings across multiple experimental approaches

  • Consider using absolute quantification methods (e.g., recombinant protein standards)

  • Account for tumor heterogeneity in tissue samples

What statistical approaches are appropriate for analyzing SMARCE1 expression in relation to clinical outcomes?

When correlating SMARCE1 expression with clinical data:

• Survival Analysis Methods:

  • Kaplan-Meier curves with log-rank tests for time-to-event outcomes

  • Cox proportional hazards models for multivariate analysis

  • Competing risk models when appropriate for cancer studies

• Expression Pattern Analysis:

  • Receiver operating characteristic (ROC) curves to determine optimal expression thresholds

  • Correlation analyses with established prognostic markers

  • Multivariate models adjusting for known prognostic factors

• Specialized Approaches:

  • Propensity score matching to control for confounding variables

  • Machine learning algorithms for complex pattern recognition

  • Meta-analysis techniques when combining multiple datasets

These approaches align with methodologies used in studies demonstrating SMARCE1's prognostic value in identifying breast tumors with metastatic potential .