EYA4 Antibody, HRP conjugated

How does EYA4 protein structure relate to antibody epitope selection?

EYA4 protein contains two functionally distinct domains that should be considered when selecting antibodies: the N-terminal variable region and the C-terminal highly conserved Eya domain (eyaHR). The eyaHR contains the phosphatase catalytic site which is essential for EYA4's enzymatic activity in dephosphorylating proteins. When selecting an EYA4 antibody, researchers should consider whether they need to detect full-length EYA4 or specific domains, particularly since truncation mutations can produce partial proteins with altered functions. Antibodies targeting the N-terminal variable region may be useful for distinguishing between different EYA family members, as this region shows greater sequence divergence. Conversely, antibodies against the C-terminal Eya domain might detect multiple EYA family proteins due to sequence conservation but are crucial for studying phosphatase activity. The interaction between EYA4 and Six1, which occurs through the eyaHR domain, is disrupted by truncating mutations that remove even small portions of the C-terminus, as demonstrated in yeast two-hybrid experiments . When designing experiments to study specific functions of EYA4, researchers should select antibodies with epitopes that do not interfere with the functional domains being studied or that specifically target these domains depending on the research question.

What are the primary applications for EYA4 antibodies in molecular and cellular research?

EYA4 antibodies have multiple applications in both basic and translational research contexts. In cancer research, they are commonly used for immunohistochemistry (IHC) to assess EYA4 expression levels in tissue microarrays, allowing correlation with clinical outcomes as demonstrated in studies of hepatocellular carcinoma where EYA4 expression served as a prognostic biomarker . For protein-protein interaction studies, EYA4 antibodies are essential for co-immunoprecipitation experiments to investigate binding partners such as Six1, which has been shown to interact with the EYA4HR domain . In cellular biology, these antibodies enable immunofluorescence studies to determine the subcellular localization of EYA4, which may shuttle between nuclear and cytoplasmic compartments depending on cellular context. For quantitative analysis of EYA4 expression, researchers employ Western blotting with EYA4 antibodies to measure protein levels in different cell lines or under various experimental conditions, such as after gene knockdown or overexpression . Additionally, chromatin immunoprecipitation (ChIP) assays using EYA4 antibodies can identify genomic regions where EYA4 functions as a transcriptional co-activator. The HRP conjugation specifically enhances detection sensitivity in applications requiring enzymatic signal amplification, such as Western blotting, ELISA, and IHC.

How can EYA4 antibodies be used to investigate its dual role in different cancer types?

EYA4 antibodies can be strategically deployed to investigate the seemingly contradictory roles of EYA4 across different cancer types. In breast cancer, where EYA4 functions as an oncogene, antibodies can be used in immunohistochemical analyses of tumor samples to correlate expression levels with tumor aggressiveness, metastatic potential, and patient outcomes. Researchers should employ quantitative image analysis software like Image-Pro Plus to objectively measure staining intensity, as was done in hepatocellular carcinoma studies . For mechanistic studies, EYA4 antibodies can be used in Western blots following EYA4 knockdown or overexpression to confirm alterations in downstream signaling pathways and to monitor changes in phosphorylation states of target proteins. In hepatocellular carcinoma, where EYA4 appears to function as a tumor suppressor, researchers can use antibodies to investigate how reduced EYA4 levels affect the c-JUN/VEGFA pathway, which has been implicated in angiogenesis and metastasis . Comparative immunoprecipitation studies across different cancer cell lines can identify tissue-specific EYA4 binding partners that might explain its opposing functions in different cancers. Additionally, phospho-specific antibodies targeting EYA4's serine/threonine and tyrosine phosphatase domains could help distinguish which enzymatic activity predominates in different cancer contexts, as the phosphatase activity appears particularly important for breast cancer progression and metastasis .

What methodologies are recommended for using EYA4 antibodies to study its role in DNA replication and genome stability?

To investigate EYA4's role in DNA replication and genome stability, researchers should implement a multi-faceted approach using HRP-conjugated EYA4 antibodies. Begin with immunofluorescence co-staining of EYA4 with γH2AX to assess whether EYA4 colocalizes with sites of DNA damage, particularly after treatment with replication stress inducers like hydroxyurea (HU). Western blotting with HRP-conjugated EYA4 antibodies should be performed alongside antibodies against DNA damage response proteins (γH2AX, pATR, pChk1) to correlate EYA4 levels with checkpoint activation states. Research has demonstrated that EYA4 depletion results in increased sensitivity to hydroxyurea and elevated levels of endogenous DNA damage indicated by increased γH2AX levels . For replication dynamics studies, combine EdU incorporation assays with EYA4 immunofluorescence to visualize how EYA4 affects DNA synthesis rates during normal replication and recovery from replication stress. Chromatin fractionation followed by Western blotting for EYA4 can determine whether it associates with chromatin during specific cell cycle phases. To assess the functional impact of EYA4 on replication fork stability, researchers should perform DNA fiber assays in cells with modulated EYA4 levels, measuring the lengths of nascent DNA strands and replication fork restart efficiency after HU treatment. These methodological approaches will provide comprehensive insights into how EYA4, particularly through its serine/threonine phosphatase domain, contributes to replication fork progression and maintains genomic stability.

What are the optimal conditions for using HRP-conjugated EYA4 antibodies in Western blotting?

For optimal detection of EYA4 using HRP-conjugated antibodies in Western blotting, researchers should implement a carefully optimized protocol that accounts for EYA4's molecular characteristics. Begin with efficient protein extraction using RIPA buffer supplemented with phosphatase inhibitors (critical since EYA4 itself has phosphatase activity) and protease inhibitors to prevent degradation. The relatively high molecular weight of EYA4 (approximately 192 kDa for the full-length protein) necessitates using lower percentage SDS-PAGE gels (6-8%) or gradient gels to achieve optimal separation. During transfer to membranes, employ longer transfer times (overnight at low voltage or 2-3 hours at higher voltage) to ensure complete transfer of these large proteins. For blocking, use 5% non-fat dry milk in TBST, but switch to 5% BSA for phospho-specific applications since milk contains phosphoproteins that may interfere with phospho-specific detection. The optimal dilution for HRP-conjugated EYA4 antibodies typically ranges from 1:1000 to 1:5000, but this should be empirically determined for each lot of antibody. Because expression levels of EYA4 can vary significantly between different tissue types (with notably different expression patterns observed between breast cancer and hepatocellular carcinoma tissues ), loading controls should be carefully selected and validated. Development with enhanced chemiluminescence (ECL) reagents optimized for HRP-conjugated antibodies will provide sensitive detection, with exposure times adjusted according to EYA4 expression levels in the experimental samples.

What considerations should be taken into account when using EYA4 antibodies for immunohistochemistry in different tissue types?

When performing immunohistochemistry with EYA4 antibodies across different tissue types, researchers must consider several technical and biological factors to obtain accurate and interpretable results. Firstly, antigen retrieval methods should be optimized specifically for EYA4 detection, with citrate buffer (pH 6.0) serving as a suitable starting point, though EDTA buffer (pH 9.0) may provide better results in some tissues. The concentration of HRP-conjugated EYA4 antibodies should be carefully titrated for each tissue type, as EYA4 expression varies considerably between tissues; for instance, EYA4 shows differential expression between hepatocellular carcinoma and breast cancer tissues . When comparing normal and pathological tissues, include internal controls within each slide and process all slides simultaneously to minimize technical variability. For quantification, instead of relying on subjective scoring, implement digital image analysis using software such as Image-Pro Plus to measure integrated optical density, as employed in HCC studies . The subcellular localization of EYA4 may vary between tissues and disease states, potentially showing both nuclear and cytoplasmic distribution, so both compartments should be evaluated separately. When studying the relationship between EYA4 and clinical outcomes, standardize the interpretation criteria—for HCC, decreased expression was defined as integrated optical density weaker in tumors than in paired non-tumorous tissue . Additionally, researchers should be aware that certain fixatives may affect phosphorylation states, which could impact the detection of EYA4 given its role as a phosphatase.

What are the best approaches for validating EYA4 antibody specificity in experimental settings?

Validating EYA4 antibody specificity is crucial for experimental rigor and requires multiple complementary approaches. First, researchers should perform knockdown validation using siRNA or shRNA targeting EYA4, followed by Western blotting to confirm reduction in the specific band corresponding to EYA4's molecular weight (approximately 192 kDa). This approach was successfully employed in studies investigating EYA4's role in breast cancer progression . For overexpression validation, transfect cells with an EYA4 expression vector containing an epitope tag (e.g., FLAG or HA) distinct from the antibody's target epitope, then perform Western blotting with both the EYA4 antibody and an antibody against the epitope tag to confirm signal overlap. Pre-absorption tests should be conducted by pre-incubating the antibody with purified EYA4 protein or the specific peptide used for immunization, which should eliminate or significantly reduce signal if the antibody is specific. When working with tissue samples, perform parallel staining with at least two different EYA4 antibodies that recognize distinct epitopes to confirm staining pattern consistency. Additionally, cross-reactivity with other EYA family members (EYA1, EYA2, and EYA3) should be assessed, particularly for antibodies targeting the more conserved C-terminal Eya domain (eyaHR). For HRP-conjugated antibodies specifically, include controls to assess non-specific binding of the secondary detection system by performing the protocol with omission of the primary antibody step.

How can researchers design experiments to distinguish between the phosphatase and transcriptional activities of EYA4?

To differentiate between EYA4's dual functions as a phosphatase and transcriptional co-activator, researchers should implement a comprehensive experimental strategy utilizing domain-specific mutations and functional assays. Begin by generating two EYA4 mutant constructs: one with mutations in the catalytic phosphatase domain that abolish enzymatic activity without affecting protein-protein interactions, and another that disrupts interactions with transcriptional partners like Six1 while preserving phosphatase activity. The interaction with Six1 occurs through the eyaHR domain and is disrupted by truncating mutations that remove even small portions of the C-terminus, as demonstrated in yeast two-hybrid experiments . Express these constructs in EYA4-depleted cells and perform rescue experiments to determine which function is required for specific cellular phenotypes. For phosphatase activity assessment, utilize in vitro phosphatase assays with purified wild-type and mutant EYA4 proteins against known substrates, or employ phospho-specific antibodies to monitor substrate phosphorylation states in cells. To evaluate transcriptional co-activator function, perform chromatin immunoprecipitation (ChIP) followed by qPCR or sequencing to identify genomic regions bound by EYA4 in complex with Six1 or other transcription factors. Additionally, use luciferase reporter assays with promoters known to be regulated by Six1-EYA4 complexes, comparing the effects of wild-type EYA4 versus phosphatase-dead or interaction-deficient mutants. These approaches will help delineate which of EYA4's functions contributes to specific cellular processes, such as its role in breast cancer progression where the serine/threonine phosphatase domain appears particularly important .

What experimental approaches can be used to investigate the relationship between EYA4 and replication stress response pathways?

To elucidate EYA4's role in replication stress response, researchers should implement a multi-layered experimental approach combining genetic, biochemical, and imaging techniques. Begin with a CRISPR-Cas9-mediated knockout or stable shRNA knockdown of EYA4, followed by complementation with wild-type or phosphatase-dead EYA4 to determine which domain is essential for replication stress response. Apply hydroxyurea (HU) treatment at various concentrations and timepoints to induce replication stress, then measure cell viability using MTT assays to assess sensitivity, as previous research showed EYA4-depleted cells are more sensitive to HU . For mechanistic insights, perform immunoprecipitation with HRP-conjugated EYA4 antibodies followed by mass spectrometry to identify novel interaction partners within the replication stress response pathway. Monitor replication dynamics using DNA fiber assays, where nascent DNA is labeled with nucleotide analogs before and after replication stress to measure fork speed, stalling, and restart efficiency in EYA4-proficient versus deficient cells. Employ proximity ligation assays (PLA) to visualize potential interactions between EYA4 and key replication stress response proteins such as ATR, RPA, or BRCA1 at sites of stalled replication forks. To assess DNA damage accumulation, quantify γH2AX foci formation after HU treatment and during recovery in the presence or absence of EYA4, as research has demonstrated that EYA4 depletion results in increased and unresolved levels of HU-induced double-strand breaks . These approaches will provide comprehensive insights into how EYA4, particularly through its serine/threonine phosphatase activity, contributes to genomic stability during replication stress.

How can researchers integrate proteomics and genomics approaches with EYA4 antibody-based studies?

Integrating proteomics and genomics with EYA4 antibody-based studies requires a systematic multi-omics approach to comprehensively understand EYA4's biological functions. Begin with chromatin immunoprecipitation followed by sequencing (ChIP-seq) using validated EYA4 antibodies to map genome-wide binding sites and identify direct transcriptional targets, particularly focusing on regions co-occupied with Six1, a known interaction partner of EYA4 . Complement this with RNA-seq analysis comparing transcriptomes of wild-type versus EYA4-depleted or overexpressing cells to identify genes whose expression is influenced by EYA4. For proteomic integration, perform immunoprecipitation with HRP-conjugated EYA4 antibodies followed by mass spectrometry (IP-MS) to identify novel EYA4 interacting proteins under normal conditions and after exposure to stressors like hydroxyurea. Additionally, conduct phosphoproteomic analysis comparing cells with normal, depleted, or phosphatase-dead EYA4 to identify substrates of EYA4's phosphatase activity, which has been shown to be critical for breast cancer progression . To connect these datasets, employ computational approaches to identify enriched pathways and biological processes affected by EYA4 modulation. For clinical relevance, correlate EYA4 ChIP-seq binding patterns or proteomics-identified interactions with patient outcome data from resources like TCGA, similar to the approach used in hepatocellular carcinoma studies where EYA4 expression served as a prognostic indicator . This integrated approach will provide a systems-level understanding of EYA4's diverse roles in normal physiology and disease states.

What are common issues encountered when using EYA4 antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with EYA4 antibodies that can be systematically addressed through careful optimization. One common issue is weak or absent signal in Western blots, which may result from low endogenous EYA4 expression in certain cell types or tissues. This can be mitigated by increasing protein loading (up to 50-100 μg per lane), extending exposure times, using more sensitive ECL substrates, or employing signal enhancement systems specifically designed for HRP-conjugated antibodies. Non-specific bands are another frequent problem, particularly when detecting endogenous EYA4 in complex samples. To address this, optimize antibody dilution (typically testing between 1:500 to 1:5000), extend blocking times, and add 0.1-0.5% Tween-20 in washing buffers to reduce background. For cross-reactivity with other EYA family members, select antibodies raised against the less conserved N-terminal variable region rather than the highly conserved C-terminal Eya domain. Variable results between experiments often stem from inconsistent transfer of high molecular weight proteins; improve reproducibility by using wet transfer systems with cooled buffers containing 10-20% methanol and extended transfer times. For immunohistochemistry applications, excessive background staining may occur; optimize by reducing antibody concentration, extending blocking steps with serum matching the host species of your secondary antibody, and including additional washing steps. If EYA4 detection remains challenging after these optimizations, consider signal amplification systems such as tyramide signal amplification (TSA) which can enhance HRP-based detection by up to 100-fold without increasing background.

How should researchers address variability in EYA4 expression levels across different experimental systems?

Addressing variability in EYA4 expression across different experimental systems requires careful methodological standardization and appropriate controls. First, establish baseline EYA4 expression levels in your model systems by quantitative RT-PCR and Western blotting before designing experiments, as EYA4 levels vary significantly between tissue types, with notably different patterns observed between breast cancer (where it can be overexpressed) and hepatocellular carcinoma (where it is often downregulated) . When comparing EYA4 expression between conditions, process all samples simultaneously using identical protocols, reagents, and imaging parameters to minimize technical variability. For Western blotting, always include positive control samples with known EYA4 expression and optimize protein loading for each cell type to prevent signal saturation or insufficiency. In immunohistochemistry applications, use tissue microarrays containing multiple samples on a single slide to eliminate batch effects, and employ automated staining platforms if available to ensure protocol consistency. When analyzing patient samples, consider creating an expression index normalized to matched normal tissues rather than using absolute values, as was done in hepatocellular carcinoma studies where EYA4 downregulation was defined relative to paired non-tumorous tissue . If EYA4 expression changes significantly with cell density or cell cycle phase, standardize culture conditions and harvesting protocols across experiments. For quantitative comparisons, use digital image analysis software rather than subjective scoring methods, employing integrated optical density measurements to objectively quantify staining intensity. These methodological precautions will minimize variability and increase reproducibility when studying EYA4 across different experimental systems.

What quality control measures should be implemented when using EYA4 antibodies for mechanistic studies?

Implementing rigorous quality control measures for EYA4 antibodies in mechanistic studies is essential for generating reproducible and reliable data. Begin with comprehensive antibody validation using multiple approaches: Western blotting with positive and negative control samples, immunoprecipitation followed by mass spectrometry to confirm target identity, and siRNA-mediated knockdown to verify signal reduction. For phospho-specific applications, treat samples with phosphatases to confirm signal loss. When investigating EYA4's role in particular pathways, such as the c-JUN/VEGFA pathway in hepatocellular carcinoma or DNA damage response in breast cancer , include pathway-specific positive controls (e.g., known pathway activators or inhibitors) to verify assay functionality. Maintain detailed records of antibody lot numbers, as lot-to-lot variability can significantly impact results, and test new lots against reference samples before use in critical experiments. For each new cell line or tissue type, optimize antibody concentration and incubation conditions, as EYA4 detection may require different parameters depending on expression level and sample complexity. When performing co-localization studies with immunofluorescence, include single-channel controls and spectral overlap corrections to ensure genuine co-localization rather than bleed-through artifacts. For mechanistic studies involving EYA4's phosphatase activity, integrate activity assays with expression analysis to correlate protein levels with functional outputs. Finally, implement blinding procedures for image acquisition and analysis to prevent unconscious bias, particularly in studies comparing EYA4 expression between normal and pathological samples or between treatment conditions.