EME1A Antibody

What is EME1 and why are antibodies against it important in research?

EME1 is a critical component of the DNA repair machinery that forms a heterodimeric endonuclease complex with MUS81. This complex contributes significantly to DNA repair processes and is associated with maintaining genomic stability . EME1 antibodies are essential research tools that allow scientists to:

• Detect and quantify EME1 protein levels in various cell types

• Investigate EME1's subcellular localization

• Study its involvement in DNA repair pathways

• Examine its role in cancer progression and genomic instability

Immunofluorescence studies have confirmed that EME1 is mainly localized in the cytoplasm, with some presence in the nucleus . This dual localization makes EME1 antibodies particularly valuable for studying the protein's differential functions across cellular compartments.

Proper validation is essential for ensuring reliable experimental results. Recommended validation approaches include:

• Western blot analysis: EME1 antibodies should detect a protein of approximately 180 kDa in cell or tissue lysates . The expression levels may vary across tissue types, with notable expression in skeletal muscle, heart, brain, lung, liver, and pancreas.

• RNA interference controls: Compare antibody signals between wildtype cells and those with EME1 knockdown. In validated studies, EME1 silencing resulted in significantly reduced signal intensity (approximately 73% knockdown efficiency in AGS cells and 69% in MGC-803 cells) .

• Immunoprecipitation verification: Test whether the antibody can effectively immunoprecipitate EME1 from cell lysates. Not all EME1 antibodies are suitable for IP applications – research has shown that only 12 out of 29 tested monoclonal antibodies could efficiently immunoprecipitate E1A polypeptides .

• Cell line panel testing: Test the antibody across multiple cell lines with known EME1 expression levels. For example, MGC-803 and AGS cell lines show particularly high EME1 expression and can serve as positive controls .

How can EME1 antibodies be used to study EME1-MUS81 complex formation?

The EME1-MUS81 endonuclease complex is central to DNA repair mechanisms. To study this complex:

• Co-immunoprecipitation (Co-IP): Use EME1 antibodies for co-IP followed by western blotting for MUS81 to assess complex formation. Research shows that EME1 variants interact with MUS81 proportionally to their abundance, indicating that antibody selection is critical for capturing physiologically relevant complexes .

• Proximity ligation assay (PLA): This technique can visualize protein-protein interactions in situ, allowing researchers to study the EME1-MUS81 complex within its native cellular environment.

• Expression of tagged variants: Studies have employed doxycycline-inducible systems expressing HA-Strep-GFP-tagged EME1 variants to investigate complex formation. Co-IP analysis revealed that wild-type EME1 (TE) and tetra-SUMO2-EME1 (T4S2E) are expressed at lower levels than mono-sumoylated EME1 (TS1E), yet all variants form functional complexes with MUS81 .

When designing such experiments, consider that post-translational modifications like sumoylation can affect complex formation – T4S2E-MUS81 interaction was observed to be reduced compared to other variants .

What role does EME1 play in cancer progression and how can antibodies help investigate this?

EME1 has been implicated in cancer progression, particularly in gastric cancer. EME1 antibodies can be instrumental in investigating its role through:

• Expression analysis in tumor tissues: Immunohistochemistry using EME1 antibodies has revealed that EME1 is upregulated in gastric cancer tissues compared to normal tissues. High EME1 expression correlates with differentiation level and lymph node metastasis .

• Functional studies: EME1 knockdown significantly reduces:

  • Colony formation

  • Cell proliferation

  • Migration and invasion capabilities

These findings suggest EME1 promotes tumorigenesis by enhancing cell proliferation and metastasis .

• Signaling pathway analysis: EME1 modulates the Akt/GSK3B/CCND1 signaling pathway. Antibodies against EME1 and these pathway components can help elucidate the mechanistic relationships. Silencing EME1 decreases expression and phosphorylation levels of Akt, CCND1, and GSK3B .

• In vivo tumor models: In xenograft models, EME1 knockdown reduces tumor growth. Immunostaining with Ki-67 antibodies shows that EME1 silencing downregulates Ki-67 expression, confirming its role in proliferation .

How does EME1 sumoylation affect its function and how can this be studied using antibodies?

EME1 sumoylation significantly impacts its function in DNA repair. Studies using cell lines expressing different sumoylated EME1 variants have revealed:

To study these relationships:

• Use specific antibodies against sumoylated proteins: These can help distinguish between different sumoylation states of EME1.

• Employ cell lines with inducible EME1 variants: Doxycycline-inducible systems expressing HA-Strep-GFP-tagged EME1 variants (wild-type, mono-sumoylated, and poly-sumoylated) allow controlled study of sumoylation effects .

• Combine with SUMO inhibitors: Pre-treatment with SUMO E1-inhibitor ML792 (1 μM) before CPT exposure helps distinguish between sumoylation-dependent and -independent functions .

• Quantitative analysis methodology: Analyze DSB formation using γH2AX staining and replication via EdU incorporation, normalizing signals to control conditions. Statistical significance should be determined using two-way ANOVA followed by Tukey test for multiple comparisons .

Research has shown that mono-sumoylation (TS1E) significantly improves double-strand break formation upon CPT exposure, while poly-sumoylation (T4S2E) enhances both DSB formation and replication .

What are the optimal conditions for EME1 antibody use in Western blotting?

For optimal Western blotting results with EME1 antibodies:

When troubleshooting:

• High background may indicate insufficient blocking or washing

• Absence of bands may suggest protein degradation or insufficient transfer

• Multiple bands may indicate protein degradation or post-translational modifications

EME1 exhibits heterogeneity in size and charge, resolving into approximately 60 polypeptide species due to both synthesis from multiple mRNAs and post-translational modifications . This heterogeneity must be considered when interpreting Western blot results.

How can I use EME1 antibodies to study its role in DNA damage response?

EME1 plays a critical role in DNA damage response. To investigate this function:

• DNA damage induction protocols:

  • Camptothecin (CPT) treatment: 10 μM for 1 hour

  • Mitomycin C (MMC): Highly effective for studying EME1 function as EME1-deficient cells show hypersensitivity to this cross-linking agent

  • Cisplatin: Another cross-linking agent that demonstrates EME1 dependency

• Experimental approach:

  • Treat cells with DNA-damaging agents with and without EME1 knockdown/knockout

  • Use EME1 antibodies to confirm knockdown efficiency

  • Measure DNA damage using γH2AX staining or comet assay

  • Assess cell viability, cell cycle distribution, and apoptosis rates

• Analytical methods:

  • Flow cytometry for cell cycle analysis and apoptosis assessment

  • Clonogenic assays for cell survival

  • Immunofluorescence microscopy for protein localization

  • Quantitative real-time PCR for expression analysis

Research has shown that EME1 deficiency leads to hypersensitivity to DNA cross-linking agents but only mild sensitivity to ionizing radiation, UV radiation, and hydroxyurea treatment .

What considerations are important when using EME1 antibodies for immunoprecipitation?

Immunoprecipitation (IP) with EME1 antibodies requires careful optimization:

• Antibody selection: Not all EME1 antibodies are suitable for IP. Research indicates that only 12 of 29 tested monoclonal antibodies efficiently immunoprecipitated E1A polypeptides from detergent lysates of infected cells .

• IP protocol optimization:

  • Cell lysis buffer: Use a buffer containing 0.2% Triton X-100 to preserve protein-protein interactions

  • Antibody amount: Typically 2-5 μg per mg of protein lysate

  • Incubation time: Overnight at 4°C for optimal antigen capture

  • Washing conditions: Multiple washes with decreasing salt concentrations

• Co-IP considerations:

  • To study EME1-MUS81 interaction, IP with EME1 antibody followed by MUS81 detection

  • Different EME1 variants (wild-type, sumoylated) interact with MUS81 at varying efficiencies

  • Expression levels affect complex formation – TE and T4S2E express at lower levels than TS1E but can still form functional complexes

• Verification of results:

  • Include proper negative controls (IgG of the same species)

  • Validate specificity by performing reverse IP when possible

  • Consider using tagged EME1 variants for confirmation using tag-specific antibodies

How are EME1 antibodies being used in cancer biomarker research?

EME1 is emerging as a potential biomarker in cancer research, particularly for gastric cancer:

• Expression analysis in tumors:

  • EME1 is upregulated in gastric cancer tissues and cell lines

  • High EME1 expression correlates with differentiation level and lymph node metastasis

  • EME1 levels may predict patient prognosis, with elevated expression associating with poorer survival

• Methodological approaches:

  • Immunohistochemistry of tissue microarrays

  • Western blot analysis of tumor samples

  • qRT-PCR for mRNA expression level quantification

• Clinical correlation studies:

  • EME1 expression can be correlated with clinicopathological characteristics

  • Survival analysis using Kaplan-Meier curves can assess prognostic value

  • Multivariate analysis can determine if EME1 is an independent prognostic factor

Research suggests that EME1 may be an important molecular marker of gastric carcinogenesis and could represent a novel candidate gene for prognosis and treatment .

What are the current limitations of EME1 antibodies and how might they be improved?

Current limitations and potential improvements for EME1 antibodies include:

• Specificity challenges:

  • EME1 exists in multiple forms due to alternative splicing and post-translational modifications

  • Current antibodies may not distinguish between these variants effectively

  • Next-generation antibodies with higher specificity for particular EME1 variants could address this limitation

• Application limitations:

  • Not all EME1 antibodies work efficiently across all applications (WB, ICC, IP)

  • Many require specific buffer conditions or may not penetrate certain cellular compartments

  • Development of application-specific antibodies could improve research outcomes

• Future improvement strategies:

  • Enhanced brain delivery systems: Recent advances in antibody delivery across the blood-brain barrier could be adapted for EME1 antibodies, potentially using biodegradable polymers like poly 2-methacryloyloxyethyl phosphorylcholine (PMPC)

  • Machine learning approaches: Computational models for antibody-antigen binding prediction could improve antibody design, as demonstrated in recent studies that reduced required antigen mutant variants by up to 35%

  • Active learning strategies: Novel active learning approaches for antibody-antigen binding prediction could advance experimental efficiency in antibody development

How can complementation studies validate EME1 antibody specificity in knockout models?

Complementation studies provide robust validation of antibody specificity and EME1 function:

• Experimental design:

  • Generate EME1 knockout cell lines or animal models

  • Reconstitute with wild-type or mutant EME1 constructs

  • Use EME1 antibodies to confirm successful reconstitution

  • Test functional recovery through phenotypic assays

• Validation methodology:

  • Express HA-tagged EME1 in EME1-/- cells and confirm expression by immunoprecipitation and western blotting

  • Test function recovery using DNA damage sensitivity assays (e.g., MMC sensitivity)

  • Quantitatively compare wild-type, knockout, and reconstituted phenotypes

• Example from research:
  In a successful complementation study, two HA-EME1 reconstituted EME1-/- ES clones showed rescue to near wild-type levels when tested for MMC sensitivity. This complementation demonstrated that the observed phenotype was caused by the specific loss of EME1, validating both the antibody specificity and the functional role of EME1 .

• Controls and considerations:

  • Include multiple reconstituted clones from different knockout parental lines

  • Use antibodies against both the endogenous protein and the tag (if applicable)

  • Ensure expression levels in reconstituted lines are physiologically relevant

What are the best practices for designing experiments with EME1 antibodies?

When designing experiments with EME1 antibodies, researchers should:

• Validate antibody specificity:

  • Test in both positive and negative control samples

  • Confirm using genetic approaches (knockdown/knockout)

  • Verify recognition of the correct molecular weight protein

• Optimize experimental conditions:

  • Determine optimal antibody concentration for each application

  • Establish appropriate incubation times and temperatures

  • Select compatible buffer systems and blocking agents

• Include proper controls:

  • Positive controls (tissues/cells known to express EME1)

  • Negative controls (EME1-knockout cells, isotype controls)

  • Loading controls for quantitative comparisons

• Consider EME1 variants and modifications:

  • Account for post-translational modifications that may affect antibody binding

  • Be aware of alternative splicing that might generate different isoforms

  • Design experiments to distinguish between various modified forms

• Data analysis recommendations:

  • Perform at least three independent experiments for statistical validity

  • Express data as mean ± SEM for quantitative analyses

  • Use appropriate statistical tests based on experimental design (e.g., two-way ANOVA followed by Tukey test for multiple comparisons)