EWSR1 Antibody

What is EWSR1 and why is it significant in cancer research?

EWSR1 (Ewing Sarcoma Breakpoint Region 1) is a multifunctional protein that plays crucial roles in preventing chromosomal instability and aneuploidy. Research has demonstrated that EWSR1 prevents aneuploidy induction through interaction with Aurora B kinase . Its significance extends beyond Ewing sarcoma, as EWSR1 knockdown has been linked to high incidence of lagging chromosomes during anaphase and aneuploidy after mitosis . When selecting antibodies for EWSR1 research, consider that the protein exists in two visual modalities in the nucleoplasm - one distributed throughout and one as discrete foci, both associated with nascent RNA .

What specific EWSR1 antibody formats are available for different research applications?

EWSR1 antibodies come in multiple formats optimized for different experimental applications:

When designing experiments, choose formats based on required applications, with polyclonal antibodies offering broader epitope recognition while monoclonals provide higher specificity .

How should I validate EWSR1 antibody specificity before experimental use?

Validation of EWSR1 antibody specificity requires a multi-step approach. Begin with Western blot analysis using both positive control cell lines (HeLa cells express detectable EWSR1 levels) and negative control samples (EWSR1 knockout/knockdown cells) . Scientific data from antibody manufacturers show that affinity-purified rabbit anti-EWSR1 antibodies detect specific bands at the expected molecular weight in human cell lysates at concentrations as low as 0.04 μg/ml . For immunoprecipitation validation, compare results between different EWSR1 antibodies (e.g., NB200-183 and NB200-182) to confirm target specificity . Additional validation should include immunofluorescence with subcellular localization confirmation, as EWSR1 displays characteristic nucleoplasmic distribution with variations in signal intensities across nuclei .

What considerations are important when selecting between monoclonal and polyclonal EWSR1 antibodies?

The selection between monoclonal and polyclonal EWSR1 antibodies depends on experimental requirements:

Polyclonal EWSR1 antibodies:

• Recognize multiple epitopes, enhancing detection sensitivity

• Often generated using synthetic peptides mapping to specific regions (e.g., residues 600-656 of human EWSR1)

• Beneficial for immunoprecipitation and initial protein characterization

• Examples include rabbit polyclonal antibodies that perform well in Western blot at 0.04 μg/ml concentrations

Monoclonal EWSR1 antibodies:

• Provide consistent lot-to-lot reproducibility

• Target specific epitopes (e.g., clone 3A9 targets AA 369-399)

• Preferred for long-term studies requiring standardized reagents

• Useful for distinguishing between wildtype EWSR1 and fusion proteins

For quantitative studies comparing EWSR1 expression across different cell lines, polyclonal antibodies may detect greater expression variation, as demonstrated in comparative analyses between A673 and TC-32 cell lines .

What are recommended protocols for EWSR1 antibody use in Western blotting?

Optimal Western blotting protocols for EWSR1 detection require careful consideration of sample preparation, antibody concentration, and detection methods:

• Sample preparation:

  • Prepare whole cell lysates using NETN lysis buffer (demonstrated effective with HeLa and NIH3T3 cells)

  • Load 15-50 μg protein per lane depending on expression levels

  • Include both human and mouse samples for cross-reactivity assessment

• Antibody incubation:

  • For affinity-purified rabbit anti-EWSR1 antibodies, use 0.04-0.1 μg/ml concentrations

  • Incubate overnight at 4°C for optimal signal-to-noise ratio

  • Include appropriate blocking buffers (typically 5% non-fat milk or BSA)

• Detection:

  • Chemiluminescence detection provides clear results with exposure times of approximately 3 seconds

  • Secondary antibody selection should match host species (anti-rabbit HRP for polyclonal rabbits antibodies)

Validation experiments have demonstrated that this protocol successfully detects EWSR1 in both human and mouse cell lines with high specificity .

How can I optimize immunoprecipitation experiments using EWSR1 antibodies?

Successful immunoprecipitation of EWSR1 requires optimization of several parameters:

• Lysate preparation:

  • Use NETN lysis buffer for cell lysis to maintain protein interactions

  • Prepare 0.5-1.0 mg protein per IP reaction

  • Clear lysates by centrifugation at high speed (>10,000g) before immunoprecipitation

• Antibody amount and incubation:

  • Use 6 μg of affinity-purified rabbit anti-EWSR1 antibody per IP reaction

  • Incubate lysate-antibody mixture overnight at 4°C with gentle rotation

  • Add protein A/G beads and continue incubation for 1-2 hours

• Washing and elution:

  • Perform stringent washing (at least 3-4 times) with lysis buffer

  • Elute immunoprecipitated proteins by boiling in SDS sample buffer

  • Load approximately 20% of IP material for subsequent Western blot detection

For detecting protein-protein interactions, such as EWSR1-Aurora B complexes, cross-linking prior to lysis may improve detection of transient interactions . When analyzing results, compare immunoprecipitation efficiency between different antibodies (e.g., NB200-183 and NB200-182) to validate specificity .

What methodologies are effective for studying EWSR1 localization using immunofluorescence?

Effective immunofluorescence studies of EWSR1 localization require specialized approaches:

• Cell preparation and fixation:

  • For optimal visualization of EWSR1's dual modalities (distributed and focal), culture cells on coverslips at 70-80% confluence

  • Fix with 4% paraformaldehyde to preserve nuclear architecture

  • Permeabilize with 0.1-0.5% Triton X-100 to allow antibody access to nuclear proteins

• Antibody selection and optimization:

  • For unmodified cells, use validated EWSR1 antibodies at optimized dilutions

  • For genetically modified systems, mNG-fused EWSR1 provides direct visualization without secondary antibody requirements

  • Include co-staining with nascent RNA markers to confirm EWSR1's association with transcriptional activities

• Advanced imaging techniques:

  • 3D-SIM (Structured Illumination Microscopy) enables high-resolution visualization of EWSR1 distribution patterns

  • Generate line plots to demonstrate variations in signal intensities across representative nuclei

  • Quantify the number of foci per central z-plane for comparative analysis between cell lines

Research has demonstrated that antibody-based immunofluorescence and direct visualization of mNG-EWSR1 fusion proteins yield comparable results for foci counting, though there may be differences in detecting expression level variations between cell lines .

How can I design experiments to investigate EWSR1's role in aneuploidy prevention?

To investigate EWSR1's role in preventing aneuploidy, a multi-faceted experimental approach is recommended:

• Conditional EWSR1 knockdown system:

  • Implement auxin-inducible degron (AID) tagging of endogenous EWSR1 to achieve controlled protein depletion

  • Confirm knockdown efficiency using Western blot and immunocytochemistry with anti-FLAG and anti-EWSR1 antibodies

  • Synchronize cells to analyze effects within a single cell cycle

• Chromosome analysis methodologies:

  • Prepare chromosome spreads using cytospin and perform immunocytochemistry with centromere markers (anti-CENPC) and chromosome visualization markers (anti-TOPO2A)

  • Score chromosome numbers across multiple cells (43-50 cells per sample, n=3 experiments)

  • Compare chromosome counts between control and EWSR1-depleted cells

• Rescue experiments with wildtype and mutant EWSR1:

  • Generate stable cell lines expressing Tet-On inducible EWSR1-mCherry or EWSR1:R565A-mCherry (mutant with reduced Aurora B interaction)

  • Integrate constructs at safe harbor AAVS1 locus using CRISPR/Cas9

  • Perform conditional knockdown of endogenous EWSR1 while inducing expression of wildtype or mutant EWSR1

This approach has successfully demonstrated that EWSR1 depletion leads to higher incidence of aberrant chromosome numbers compared to control cells, and that the interaction between EWSR1 and Aurora B is critical for preventing aneuploidy .

What approaches best characterize the distinct visual modalities of EWSR1 in the nucleoplasm?

Characterization of EWSR1's visual modalities requires specialized imaging and analytical techniques:

• Complementary visualization methods:

  • Compare antibody-based detection with genetically-encoded fluorescent tags (mNG-EWSR1)

  • Employ 3D-SIM for high-resolution imaging of nucleoplasmic distribution

  • Generate line plots across representative nuclei to quantify signal intensity variations

• Quantitative analysis:

  • Measure the number and size of EWSR1 foci per central z-plane

  • Compare foci characteristics between different cell lines (e.g., A673 vs. TC-32)

  • Correlate EWSR1 expression levels (determined by flow cytometry or immunoblot) with visual patterns

• Functional correlations:

  • Co-localize EWSR1 with nascent RNA to establish functional relevance of different modalities

  • Track changes in EWSR1 distribution patterns during cell cycle progression

  • Analyze how mutations affect distribution between diffuse and focal patterns

Research has shown that while antibody-based methods and fluorescent protein tagging both detect EWSR1 foci consistently, antibody-based methods may be less effective at reflecting cell-line specific differences in expression levels compared to direct visualization of tagged proteins .

How should I analyze contradictory results in EWSR1 antibody-based experiments?

When faced with contradictory results in EWSR1 antibody experiments, implement the following analytical framework:

• Antibody validation assessment:

  • Verify antibody specificity using multiple techniques (Western blot, immunoprecipitation, immunofluorescence)

  • Compare results across different antibodies targeting distinct epitopes of EWSR1

  • Ensure proper controls are included (positive controls, negative controls, EWSR1 knockdown/knockout samples)

• Cell type and context considerations:

  • Compare EWSR1 expression and localization across different cell lines (e.g., A673 vs. TC-32)

  • Consider cell-specific differences in expression levels that may affect detection sensitivity

  • Analyze how cell cycle stage affects EWSR1 distribution and function

• Methodological refinement:

  • Test different fixation and permeabilization protocols for immunofluorescence studies

  • Optimize antibody concentrations based on expression levels in specific cell types

  • Implement alternative detection methods (chemiluminescence vs. fluorescence-based detection)

• Biological interpretation:

  • Consider that EWSR1 exists in different modalities (distributed vs. focal) that may be differentially detected

  • Analyze whether EWSR1 fusion proteins in certain cell types may affect antibody recognition

  • Evaluate whether post-translational modifications impact antibody binding

Comprehensive evaluation using this framework will help resolve contradictions and provide more reliable experimental outcomes.

How can EWSR1 antibodies be used to investigate chromosomal instability mechanisms?

EWSR1 antibodies offer powerful tools for investigating chromosomal instability mechanisms through several advanced applications:

• Co-immunoprecipitation studies:

  • Use EWSR1 antibodies (6 μg per reaction) to immunoprecipitate protein complexes

  • Identify interaction partners involved in chromosome segregation, particularly Aurora B

  • Analyze how mutations (e.g., R565A) affect protein-protein interactions critical for chromosomal stability

• Microscopy-based assays:

  • Implement immunofluorescence to track Aurora B localization at inner centromeres and kinetochore-proximal chromosomes during prophase/metaphase

  • Quantify lagging chromosomes during anaphase following EWSR1 depletion

  • Correlate EWSR1 localization patterns with specific phases of mitosis

• Functional genomics approaches:

  • Combine EWSR1 antibodies with genomic techniques to map EWSR1 binding sites on chromosomes

  • Correlate EWSR1 genomic binding with chromosomal stability markers

  • Investigate how EWSR1 fusion proteins affect genomic stability mechanisms

Research has demonstrated that EWSR1 depletion for one cell cycle is sufficient to evict Aurora B from inner centromeres, enrich it at kinetochore-proximal chromosomes during pro/metaphase, and induce a high incidence of lagging chromosomes during anaphase . These findings highlight EWSR1's critical role in error correction during mitosis and suggest that cells lacking EWSR1 may develop chromosomal instability by overriding error correction processes .

What methodological approaches can distinguish between wildtype EWSR1 and fusion proteins?

Distinguishing between wildtype EWSR1 and fusion proteins requires specialized methodological approaches:

• Epitope-specific antibody selection:

  • Choose antibodies targeting N-terminal regions to detect both wildtype EWSR1 and fusion proteins

  • Select C-terminal targeting antibodies (e.g., residues 600-656) to specifically detect wildtype EWSR1

  • Compare staining patterns between N- and C-terminal antibodies to identify fusion protein presence

• Western blot analysis:

  • Run protein samples on gradient gels (4-12%) to effectively separate proteins of different molecular weights

  • Compare band patterns between wildtype EWSR1 (68 kDa) and fusion proteins (variable MW depending on fusion partner)

  • Use dual-color Western blot with antibodies against both EWSR1 and common fusion partners

• Immunofluorescence pattern analysis:

  • Compare subcellular localization patterns between wildtype and fusion proteins

  • Analyze nucleoplasmic distribution patterns and foci characteristics

  • Perform co-localization studies with markers of specific nuclear compartments

• Genetic tagging strategies:

  • Implement CRISPR/Cas9 to tag endogenous EWSR1 with fluorescent proteins

  • Monitor localization and dynamics of tagged proteins in live cells

  • Compare fusion protein function with wildtype using rescue experiments in EWSR1-depleted cells

These approaches enable researchers to distinguish between wildtype EWSR1 and fusion proteins, which is particularly important in studies of Ewing sarcoma and other EWSR1-fusion associated cancers.

How can I design experiments to investigate the relationship between EWSR1 and RNA processing?

To investigate EWSR1's role in RNA processing, consider the following experimental design approaches:

• Co-localization studies:

  • Perform dual immunofluorescence with EWSR1 antibodies and markers of nascent RNA

  • Analyze overlap between EWSR1 modalities (distributed and focal) and RNA processing factors

  • Implement super-resolution microscopy (3D-SIM) for detailed spatial relationship analysis

• RNA-protein interaction assays:

  • Combine EWSR1 immunoprecipitation with RNA sequencing to identify bound transcripts

  • Perform CLIP-seq (Cross-linking immunoprecipitation sequencing) using optimized EWSR1 antibodies

  • Compare RNA binding profiles between wildtype EWSR1 and functional mutants

• Functional depletion studies:

  • Implement AID-tagging for conditional EWSR1 depletion

  • Analyze changes in RNA processing, splicing patterns, and transcriptome profiles

  • Perform rescue experiments with domain-specific EWSR1 mutants to identify regions critical for RNA processing

• Live-cell imaging:

  • Generate cell lines expressing fluorescently tagged EWSR1 (e.g., mNG-EWSR1)

  • Track dynamics of EWSR1 in relation to nascent transcription sites

  • Analyze how perturbations in transcription affect EWSR1 localization patterns

Research has established that both distributed and focal EWSR1 modalities localize with nascent RNA , suggesting important functional relationships between EWSR1 and RNA processing mechanisms that warrant further investigation.

What are the most common issues when using EWSR1 antibodies and how can they be resolved?

When working with EWSR1 antibodies, researchers commonly encounter several issues that can be effectively addressed:

• Non-specific binding and background:

  • Problem: High background signal in Western blots or immunofluorescence

  • Solution: Increase blocking time/concentration (5% BSA or non-fat milk), optimize antibody dilution (start with manufacturer-recommended 0.04-0.1 μg/ml) , and increase washing steps

• Variable immunofluorescence patterns:

  • Problem: Inconsistent visualization of EWSR1's dual modalities (distributed vs. focal)

  • Solution: Optimize fixation protocols, use super-resolution microscopy techniques like 3D-SIM , and ensure proper z-stack acquisition to capture all EWSR1 foci

• Immunoprecipitation efficiency:

  • Problem: Poor recovery of EWSR1 or interacting partners

  • Solution: Increase antibody amount (6 μg per reaction has been validated) , optimize lysis conditions (NETN buffer works effectively) , and consider cross-linking for transient interactions

• Antibody batch variation:

  • Problem: Inconsistent results between antibody lots

  • Solution: Perform side-by-side validation of new lots against previously validated lots, maintain reference samples, and consider monoclonal antibodies for critical applications requiring consistent performance

• Detection sensitivity:

  • Problem: Weak signal in low-expressing samples

  • Solution: Use signal amplification methods, increase protein loading (up to 50 μg for Western blot) , and consider more sensitive detection reagents

Proper experimental design with appropriate controls and optimization for specific applications will resolve most common issues encountered with EWSR1 antibodies.

What controls should be included in EWSR1 antibody-based experiments?

Comprehensive controls are essential for reliable EWSR1 antibody experiments:

• Positive controls:

  • Cell lines with confirmed EWSR1 expression (HeLa cells have been validated)

  • Recombinant EWSR1 protein standards for quantitative analyses

  • EWSR1-overexpressing cells for sensitivity assessment

• Negative controls:

  • EWSR1 knockdown or knockout samples using AID-tagging systems or CRISPR/Cas9

  • Isotype control antibodies to assess non-specific binding

  • Secondary antibody-only controls for immunofluorescence and flow cytometry

• Specificity controls:

  • Peptide competition assays using the immunizing peptide (e.g., synthetic peptide mapping to residues 600-656)

  • Comparison between different antibodies targeting distinct EWSR1 epitopes

  • Western blot analysis to confirm detection of the correct molecular weight protein

• Experimental validation controls:

  • For functional studies, include rescue experiments with wildtype EWSR1 and mutant variants (e.g., EWSR1:R565A)

  • Use multiple detection methods to confirm findings (e.g., both Western blot and immunofluorescence)

  • Compare antibody-based detection with direct visualization using fluorescently tagged EWSR1

Implementing these controls ensures experimental reliability and facilitates troubleshooting when unexpected results occur.

How do I determine the optimal EWSR1 antibody concentration for different applications?

Determining optimal EWSR1 antibody concentrations requires systematic titration for each application:

• Western blot optimization:

  • Starting range: 0.04-0.1 μg/ml based on validated protocols

  • Perform titration series (e.g., 0.01, 0.05, 0.1, 0.5, 1.0 μg/ml)

  • Evaluate signal-to-background ratio at each concentration

  • Select concentration that provides clear specific bands with minimal background

• Immunofluorescence optimization:

  • Initial dilution based on manufacturer recommendations

  • Prepare serial dilutions across 3-5 concentrations

  • Score results based on signal intensity, specificity of nucleoplasmic distribution, and foci clarity

  • Consider cell type differences, as optimal concentrations may vary between cell lines with different EWSR1 expression levels

• Immunoprecipitation optimization:

  • Test range from 1-10 μg antibody per reaction (6 μg has been validated)

  • Analyze recovery efficiency at different antibody amounts

  • Consider protein expression levels in sample (higher antibody amounts for lower expression)

  • Optimize antibody-to-lysate ratio for maximum specific recovery

• Flow cytometry optimization:

  • Begin with 1:100 dilution and test serial dilutions

  • Analyze median fluorescence intensity and separation from negative controls

  • Determine saturation point where increased antibody concentration no longer improves signal

Optimal concentrations should be determined empirically for each cell line, application, and specific EWSR1 antibody used in the study.