emsy Antibody

What is EMSY and what cellular functions does it regulate?

EMSY is a nuclear protein that binds to the BRCA2 N-terminal domain and functions as a transcriptional repressor that links the BRCA2 pathway to sporadic breast and ovarian cancers . Recent research has revealed that EMSY plays critical roles in inhibiting homologous recombination repair (HRR) and the interferon response, which promotes lung cancer immune evasion . EMSY accumulation is responsible for the BRCAness phenotype observed in KEAP1-deficient cells, as it impairs the formation of RAD51 foci following genotoxic stress while not affecting pH2ax foci formation . This suggests EMSY specifically impacts HRR without disrupting initial DNA damage recognition processes. Additionally, EMSY has been implicated in tumor growth regulation, as gene set enrichment analysis showed that EMSY activity correlates with downregulation of both DNA double-strand break repair and homologous recombination repair pathways in tumor models .

Which experimental techniques are most suitable for EMSY protein detection?

Several techniques have been validated for EMSY detection, with Western Blot (WB), Immunocytochemistry/Immunofluorescence (ICC/IF), and Immunohistochemistry (IHC) being the most common applications . For protein expression analysis in cell and tissue lysates, Western Blot remains the gold standard, requiring approximately 50 micrograms of protein extract separated on 8% sodium dodecyl sulfate-polyacrylamide gel, followed by transfer to nitrocellulose membranes and probing with anti-EMSY antibodies (typically at 1/3,000 dilution for crude rabbit antisera) . For subcellular localization studies, ICC/IF provides superior resolution to visualize nuclear localization patterns typical of EMSY. In tissue sections, IHC allows researchers to assess EMSY expression patterns across different cell types within the tumor microenvironment, which is particularly valuable for studies investigating heterogeneity of EMSY expression in cancer samples .

What are the available species reactivities for EMSY antibodies and how should this guide experimental design?

The selection of an appropriate EMSY antibody depends critically on the experimental model system. Based on current commercial offerings, researchers have access to antibodies with the following reactivity profiles:

When designing cross-species experiments, researchers should prioritize antibodies with confirmed reactivity across the species of interest . For translational studies comparing human samples with mouse models, antibodies A91953 or A9800 would be appropriate. For human-specific applications requiring IHC, the A31118 antibody offers the necessary specificity and application versatility . When working with rat models, options are more limited, with only A91953 showing validated reactivity, which may necessitate additional optimization steps when using this antibody in rat-based studies.

How should EMSY antibodies be validated before use in critical experiments?

Proper validation of EMSY antibodies is essential to ensure experimental reliability. A comprehensive validation approach should include:

• Positive and negative controls: Use cell lines with known EMSY expression levels. For example, MCF-7 breast cancer cells have been validated for EMSY expression studies . Include EMSY-knockout or EMSY-silenced cells as negative controls.

• Specificity verification: Test antibody specificity using lysates from cells expressing wild-type EMSY versus mutant variants, such as the Emsy(EEGE/AAAA) mutant that cannot bind to KEAP1 . The antibody should detect differences in expression levels between these variants.

• Cross-reactivity assessment: When working with multiple species, validate the antibody in each species system separately before conducting comparative studies.

• Detection of expected molecular weight: Confirm that the antibody detects a protein of the expected molecular weight (~140 kDa for human EMSY) by Western blot.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm that specific binding is abolished, which demonstrates antibody specificity.

• Multiple detection methods: Validate the antibody across multiple techniques (WB, IF, IHC) to ensure consistent results across platforms .

How can EMSY antibodies be used to investigate the interplay between KEAP1 and EMSY in cancer models?

EMSY antibodies are crucial tools for investigating the regulatory relationship between KEAP1 and EMSY in cancer models, particularly in non-small cell lung cancers (NSCLCs). Recent research has revealed that KEAP1 targets EMSY for ubiquitin-mediated degradation, thereby regulating homologous recombination repair and anti-tumor immunity .

To investigate this relationship, researchers can employ several methodological approaches using EMSY antibodies:

• Quantitative analysis of EMSY levels: Western blotting with anti-EMSY antibodies can be used to measure EMSY protein levels in KEAP1 wild-type versus KEAP1-deficient cancer cells, which should reveal increased EMSY accumulation in KEAP1-deficient settings .

• Investigation of protein stability: Researchers can perform cycloheximide chase assays (blocking new protein synthesis) in both KEAP1-proficient and KEAP1-deficient cells, then use EMSY antibodies to track protein degradation rates over time via Western blot.

• Assessment of oxidative stress response: Following induction of oxidative stress, EMSY antibodies can demonstrate that wild-type EMSY levels oscillate while mutant Emsy(EEGE/AAAA) levels remain stable, as this mutant cannot bind KEAP1 and is therefore insensitive to KEAP1-mediated degradation .

• Co-immunoprecipitation studies: EMSY antibodies can be used to pull down EMSY complexes from cell lysates, followed by Western blotting for KEAP1 to confirm their direct interaction in vivo. This approach can be combined with mutation studies of the KEAP1 binding motif in EMSY (such as the EEGE motif at positions 871-874) .

• Immunofluorescence co-localization: Dual staining with EMSY and KEAP1 antibodies can reveal their subcellular co-localization patterns and how these change under different conditions like genotoxic stress.

How do EMSY antibodies contribute to understanding the BRCAness phenotype in cancer cells?

EMSY antibodies play a critical role in researching the BRCAness phenotype, which refers to characteristics of BRCA1/2-deficient tumors that appear in cancers without BRCA1/2 mutations. For EMSY-related BRCAness investigations, researchers can implement these methodological approaches:

• EMSY expression correlation with PARP inhibitor sensitivity: Using EMSY antibodies for immunoblotting, researchers can quantify EMSY levels across cancer cell lines and correlate expression with sensitivity to PARP inhibitors (PARPi). EMSY overexpression has been shown to disrupt the BRCA2/RAD51 pathway , and cells with EMSY accumulation display impaired growth when treated with PARPi, similar to BRCA-deficient cells .

• Assessment of homologous recombination repair deficiency: Following induction of genotoxic stress, EMSY antibodies used in immunofluorescence can help quantify RAD51 foci formation. In cells with EMSY accumulation (such as those expressing the KEAP1-binding deficient mutant Emsy(EEGE/AAAA)), RAD51 foci formation is impaired despite normal pH2ax foci formation, indicating a specific defect in homologous recombination repair rather than in DNA damage sensing .

• In vivo tumor growth studies: EMSY antibodies can be used for immunohistochemical analysis of xenograft tumors to correlate EMSY expression with tumor growth characteristics and response to therapy. This approach revealed that subcutaneously transplanted tumors expressing Emsy(EEGE/AAAA) displayed impaired growth upon PARPi treatment, similar to KEAP1-deficient tumors .

• Gene expression correlation studies: Combining EMSY antibody-based protein quantification with RNA-seq transcriptional profiling allows researchers to correlate EMSY protein levels with gene expression patterns. Gene set enrichment analysis revealed downregulation of both DNA double-strand break repair and homologous recombination repair pathways in tumors with high EMSY expression .

What technical considerations are critical when using EMSY antibodies in DNA repair pathway research?

DNA repair pathway research using EMSY antibodies requires careful technical considerations to ensure reliable results:

• Experimental timing: When studying DNA repair kinetics, precise timing of fixation after DNA damage induction is critical. For RAD51 foci detection using EMSY and RAD51 antibodies, researchers should establish a time-course experiment to determine the optimal time points, as foci formation is dynamic .

• Appropriate damage induction: The method of DNA damage induction should be selected based on the specific DNA repair pathway being studied. For homologous recombination studies involving EMSY, double-strand breaks can be induced using ionizing radiation, etoposide, or site-specific endonucleases like I-SceI .

• Quantification methodology: For foci quantification in immunofluorescence experiments, establish clear criteria for what constitutes a focus and use automated image analysis software to reduce subjective bias. When counting RAD51 foci in cells with different EMSY expression levels, consistent thresholds must be applied .

• Controls for recombination assays: When using recombination reporter constructs (such as the direct-repeat or inverted-repeat systems described in result 5), appropriate controls should include cells with known recombination proficiency and deficiency. These reporter systems typically contain inactive PuroR genes that become functional through recombination/repair events, allowing for colony assay quantification .

• Validating antibody specificity in the context of DNA damage: EMSY antibodies should be tested for specificity in both undamaged and damaged conditions, as some epitopes may become masked or modified following DNA damage induction.

How can researchers optimize EMSY antibody use for immunoprecipitation studies of protein-protein interactions?

Optimizing immunoprecipitation (IP) protocols for studying EMSY interactions requires attention to several methodological details:

• Antibody selection: Choose an anti-EMSY antibody that has been validated for IP applications. Not all antibodies that work well in Western blot or immunofluorescence will perform adequately in IP due to differences in epitope accessibility in native conditions.

• Cross-linking consideration: For transient or weak interactions, consider using membrane-permeable crosslinking agents before cell lysis to stabilize protein complexes. This may be particularly important for capturing dynamic interactions between EMSY and its binding partners during DNA damage response.

• Lysis buffer optimization: The composition of lysis buffer significantly impacts IP efficiency. For EMSY interactions, use buffers that maintain nuclear protein solubility while preserving protein-protein interactions. Buffers containing 150-300 mM NaCl, 1% NP-40 or Triton X-100, with phosphatase and protease inhibitors are typically suitable starting points.

• Pre-clearing step: To reduce non-specific binding, pre-clear lysates with protein A/G beads before adding the EMSY antibody. This is particularly important when studying interactions with abundant proteins that might bind non-specifically.

• Bead selection: Choose appropriate beads based on the host species of your EMSY antibody. For rabbit polyclonal EMSY antibodies (which are common according to search result 4), protein A or protein A/G beads are typically most effective.

• Elution conditions: Optimize elution conditions to efficiently release EMSY complexes without contaminating the sample with antibody heavy and light chains. Consider using non-reducing conditions if studying interactions that might be sensitive to reducing agents.

• Validation with reverse IP: Confirm interactions identified using EMSY antibodies by performing reverse immunoprecipitation with antibodies against the putative interaction partners.

What approaches can improve antibody-based detection of low-abundance EMSY in clinical samples?

Detection of low-abundance EMSY in clinical samples presents significant challenges that can be addressed through several methodological approaches:

• Signal amplification systems: For immunohistochemistry applications, employ tyramide signal amplification (TSA) or polymer-based detection systems to enhance sensitivity when detecting low EMSY levels in tissue sections.

• Optimized antigen retrieval: For formalin-fixed, paraffin-embedded samples, test multiple antigen retrieval methods (heat-induced epitope retrieval with citrate buffer at pH 6.0 versus EDTA buffer at pH 9.0) to determine which best exposes EMSY epitopes for antibody binding.

• Extended primary antibody incubation: For low-abundance targets, extending primary antibody incubation to overnight at 4°C often improves sensitivity compared to shorter incubations at room temperature.

• Sample enrichment techniques: For protein lysates, consider immunoprecipitation with EMSY antibodies as an enrichment step before Western blotting to concentrate the target protein.

• Proximity ligation assay (PLA): This technique can detect protein-protein interactions with single-molecule sensitivity, making it valuable for detecting low-abundance EMSY interactions in tissue samples.

• Careful validation against positive controls: Use cell lines with confirmed EMSY expression as positive controls, ideally processing these controls alongside clinical samples to enable direct comparison of signal intensity.

• Quantitative image analysis: Employ digital image analysis with appropriate software to quantify low-level immunohistochemical staining that might be difficult to assess visually, enabling more objective assessment of EMSY expression patterns across different samples.

How should researchers design experiments to study EMSY's role in immune evasion mechanisms?

Investigating EMSY's role in immune evasion mechanisms requires careful experimental design integrating both in vitro and in vivo approaches:

• Gene expression profiling: Researchers should compare transcriptional profiles of EMSY-high versus EMSY-low tumors using RNA-seq. Gene set enrichment analysis has revealed that EMSY overexpression correlates with downregulation of interferon (IFN) signature genes . Design experiments to validate these findings through qRT-PCR of key interferon-stimulated genes.

• Immune cell infiltration analysis: Design immunohistochemistry panels using EMSY antibodies alongside markers for various immune cell populations (CD8+ T cells, NK cells, macrophages) to correlate EMSY expression with immune infiltration patterns in tissue sections.

• Functional immune assays: Establish co-culture systems of cancer cells with varying EMSY expression levels and immune effector cells to measure functional outcomes like T-cell activation, proliferation, and cytotoxicity against target cells.

• EMSY silencing experiments: Design studies comparing immune responses against EMSY-high versus EMSY-silenced tumors. RNA-seq data has shown significant enrichment of interferon and innate immune gene signatures upon EMSY silencing in certain tumor models , suggesting that EMSY knockdown may enhance anti-tumor immunity.

• Cytokine profiling: Measure secreted cytokines in the supernatants of EMSY-modulated cells to determine how EMSY expression affects the inflammatory microenvironment.

• In vivo tumor models: Design syngeneic mouse models with tumors expressing either wild-type EMSY or EMSY with mutations affecting its binding to KEAP1, and compare tumor growth rates, immune infiltration, and response to immunotherapies.

What considerations are important when developing quantitative assays for measuring EMSY protein levels?

Developing robust quantitative assays for EMSY requires addressing several methodological challenges:

• Standard curve establishment: For absolute quantification, generate recombinant EMSY protein standards at known concentrations to create standard curves. Include these standards on each assay run to enable conversion of signal intensity to protein concentration.

• Sample preparation standardization: Implement consistent cell lysis protocols with specific buffer-to-cell ratios to ensure comparable protein extraction efficiency across samples. This is particularly important when comparing EMSY levels between different cell lines or tissue types.

• Loading control selection: Choose appropriate loading controls based on the experimental context. For studies involving oxidative stress or DNA damage, traditional housekeeping proteins like GAPDH or β-actin may be affected, so consider alternative loading controls like total protein staining methods (Ponceau S, SYPRO Ruby).

• Antibody validation for quantification: Verify the linear range of antibody detection by performing serial dilutions of positive control samples. Confirm that signal intensity correlates linearly with protein concentration within the expected range of EMSY expression in experimental samples.

• Digital image acquisition parameters: For Western blot quantification, establish standardized image acquisition settings to avoid saturation of high-intensity bands, which would invalidate quantitative comparisons. Use 16-bit image depth for greater dynamic range.

• Technical replicates: Include at least three technical replicates for each biological sample to account for variability in the assay itself.

• ELISA development considerations: When developing sandwich ELISA for EMSY quantification, carefully select capture and detection antibodies that recognize non-overlapping epitopes to maximize sensitivity and specificity.

How can researchers address common challenges with antibody specificity when studying EMSY?

Addressing specificity challenges when using EMSY antibodies requires systematic troubleshooting:

• Cross-reactivity mapping: If non-specific bands appear in Western blots, perform peptide competition assays with the immunizing peptide to distinguish specific from non-specific signals. Additionally, test the antibody on lysates from cells with EMSY knockdown or knockout to identify which bands represent true EMSY signal.

• Epitope masking considerations: If EMSY detection varies across experimental conditions, consider whether post-translational modifications or protein-protein interactions might be masking the epitope. Test multiple antibodies targeting different regions of EMSY to overcome this limitation.

• Isoform-specific detection: Human EMSY has multiple isoforms due to alternative splicing. Determine which isoforms are recognized by your antibody by comparing the epitope sequence with known isoform sequences. This is particularly important when comparing EMSY detection across different tissue types or species.

• Batch-to-batch variation: Polyclonal antibodies may show batch-to-batch variation in specificity and sensitivity. When receiving a new lot of EMSY antibody, perform side-by-side comparisons with the previous lot using positive control samples to calibrate working concentrations.

• Species-specific optimization: When using antibodies across species, optimize conditions separately for each species. An antibody working at 1:3000 dilution for human samples might require different dilutions for mouse or rat samples .

• Non-specific binding in immunofluorescence: If high background occurs in immunofluorescence applications, optimize blocking conditions (try different blocking agents like BSA, normal serum, or commercial blockers) and include an additional permeabilization step to improve nuclear antigen accessibility for EMSY detection.

What are the best practices for long-term storage and handling of EMSY antibodies to maintain activity?

Proper storage and handling of EMSY antibodies is critical for maintaining their activity and ensuring reproducible results:

• Aliquoting strategy: Upon receiving a new antibody, divide it into small single-use aliquots (typically 10-20 μL) to minimize freeze-thaw cycles, which can denature antibody proteins and reduce activity. Each freeze-thaw cycle can decrease antibody activity by approximately 5-10%.

• Storage temperature selection: Store antibody aliquots at -20°C or -80°C for long-term storage. While -20°C is sufficient for most antibodies, -80°C may provide better preservation of activity for extended periods (>1 year).

• Working dilution storage: Diluted working solutions of EMSY antibodies can be stored at 4°C with preservatives (0.02% sodium azide) for up to 2 weeks, but prepare fresh dilutions for critical experiments.

• Contaminant prevention: Use sterile technique when handling antibody solutions to prevent microbial contamination. Add sodium azide (0.02%) to antibody dilutions that will be stored for more than 24 hours at 4°C.

• Carrier protein consideration: For very dilute antibody solutions, consider adding carrier proteins like BSA (0.1-0.5%) to prevent antibody adsorption to storage vessels and stabilize the antibody.

• Temperature transition management: When removing antibodies from freezer storage, allow them to thaw completely at 4°C rather than at room temperature to minimize protein denaturation from rapid temperature changes.

• Documentation practices: Maintain detailed records of antibody performance across different experiments to track any potential decline in activity over time, which may indicate storage-related degradation.

How might advances in antibody development technologies improve EMSY research?

Emerging antibody technologies offer significant potential to advance EMSY research:

• Rational antibody design approaches: Computational methods like the in silico solubility predictor CamSol can be applied to antibody development, allowing researchers to design antibodies with improved solubility and reduced aggregation potential . This approach could lead to more stable and reliable EMSY antibodies for challenging applications.

• Single-domain antibodies: Nanobodies (single-domain antibodies derived from camelids) offer advantages for detecting EMSY in structurally complex environments due to their small size (~15 kDa compared to ~150 kDa for conventional antibodies). Their reduced size may improve access to sterically hindered epitopes within chromatin-bound EMSY complexes.

• Bispecific antibodies: Developing bispecific antibodies that simultaneously target EMSY and its interaction partners (like BRCA2 or KEAP1) could enable direct visualization of protein complexes in situ, providing spatial information about EMSY interactions in different cellular compartments.

• Epitope-specific recombinant antibodies: Phage display technology enables selection of recombinant antibodies against specific EMSY epitopes, including post-translationally modified regions. This could lead to antibodies that specifically recognize phosphorylated, ubiquitinated, or otherwise modified EMSY forms.

• Diffusion model-based antibody design: Recent advances in diffusion probabilistic models for antibody design represent a promising approach for developing antibodies with high specificity and affinity . These models could be applied to create EMSY antibodies optimized for specific applications like super-resolution microscopy.

• Complementarity-determining region (CDR) optimization: Force-guided sampling in diffusion models offers potential for designing antibodies with CDRs specifically optimized for EMSY detection in challenging interfaces . This could be particularly valuable for developing antibodies that can distinguish between EMSY in different conformational states.

What emerging applications of EMSY antibodies might open new research avenues?

Several emerging applications of EMSY antibodies could significantly expand research possibilities:

• Proximity-based labeling techniques: Combining EMSY antibodies with proximity labeling enzymes (APEX2, BioID, TurboID) could enable comprehensive mapping of the EMSY interactome in different cellular contexts and under various stress conditions.

• Live-cell imaging applications: Developing cell-permeable EMSY antibody fragments conjugated to fluorescent reporters could enable real-time tracking of EMSY dynamics during DNA damage response and repair processes.

• Single-cell protein profiling: Using EMSY antibodies in mass cytometry (CyTOF) or single-cell Western blotting could reveal heterogeneity in EMSY expression and activation states across individual cells within tumors, potentially identifying subpopulations with distinct therapeutic vulnerabilities.

• Antibody-guided CRISPR screening: EMSY antibodies could be used to isolate cells with particular EMSY expression patterns or modifications, which could then be subjected to CRISPR screening to identify synthetic lethal interactions specific to EMSY-overexpressing cells.

• Therapeutic targeting strategies: Knowledge gained from EMSY antibody research could inform the development of therapeutic approaches targeting the EMSY pathway, particularly in cancers showing EMSY-mediated immune evasion. This could include developing antibody-drug conjugates or proteolysis-targeting chimeras (PROTACs) directed against EMSY.

• Conformational state-specific antibodies: Developing antibodies that specifically recognize EMSY in different conformational states (e.g., BRCA2-bound versus unbound) could provide insights into how EMSY's structural dynamics relate to its various functions in transcriptional regulation and DNA repair.