EYA4 Antibody, Biotin conjugated

What is EYA4 and why is it an important research target?

EYA4 (Eyes Absent Homolog 4) is a member of the EYA protein family that functions as a transcriptional coactivator and tyrosine phosphatase. It specifically dephosphorylates 'Tyr-142' of histone H2AX (H2AXY142ph) and is highly expressed in heart and skeletal muscle tissue . Research has demonstrated that EYA4 plays critical roles in various cellular processes including cell proliferation and mitotic regulation through PLK1 activation . The protein has significant relevance in both developmental biology and disease pathology, particularly in cancer research where EYA proteins have been identified as tumor-promoting across multiple cancer types .

What are the specific characteristics of biotin-conjugated EYA4 antibody (ABIN1701247)?

The biotin-conjugated EYA4 antibody (ABIN1701247) is a rabbit polyclonal antibody that targets amino acids 301-400 of human EYA4 protein. This antibody is purified using Protein A chromatography and is generated using a KLH-conjugated synthetic peptide derived from human EYA4 . The liquid formulation contains 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol . The biotin conjugation allows for enhanced detection sensitivity when used with streptavidin-based detection systems in various immunoassays.

What are the primary applications for biotin-conjugated EYA4 antibody?

The biotin-conjugated EYA4 antibody (ABIN1701247) is validated for several applications:

• ELISA (Enzyme-Linked Immunosorbent Assay)

• Immunohistochemistry on paraffin-embedded sections (IHC-P) with recommended dilutions of 1:200-400

• Immunohistochemistry on frozen sections (IHC-F) with recommended dilutions of 1:100-500

While not specifically validated for Western blotting, other EYA4 antibodies have shown efficacy in this application at dilutions ranging from 1:500-1:3000 .

What is the optimal protocol for using biotin-conjugated EYA4 antibody in immunohistochemistry?

For optimal results in immunohistochemistry applications with biotin-conjugated EYA4 antibody, the following methodological approach is recommended:

• Tissue preparation: Fix tissues in 10% neutral buffered formalin and embed in paraffin. Cut sections at 4-6 μm thickness.

• Antigen retrieval: Based on data from other EYA4 antibodies, TE buffer pH 9.0 is recommended for antigen retrieval, though citrate buffer pH 6.0 may serve as an alternative .

• Blocking: Block endogenous peroxidase with 3% H₂O₂ and prevent non-specific binding with appropriate blocking buffer containing serum from the same species as the secondary antibody.

• Primary antibody incubation: Apply biotin-conjugated EYA4 antibody at the recommended dilution (1:200-400 for IHC-P, 1:100-500 for IHC-F) and incubate overnight at 4°C.

• Detection: Since the antibody is already biotin-conjugated, proceed directly with streptavidin-HRP. Develop signal using an appropriate chromogen such as DAB.

• Counterstaining: Counterstain with hematoxylin, dehydrate, and mount.

For validation purposes, include appropriate positive controls such as heart or skeletal muscle tissue, which express high levels of EYA4 .

How should researchers optimize Western blot protocols when using EYA4 antibodies?

While the specific biotin-conjugated antibody (ABIN1701247) is not explicitly validated for Western blot, related EYA4 antibodies can be used effectively. Based on published protocols, the following methodology is recommended:

• Sample preparation: Lyse cells in cold lysis buffer, then resolve 20-30 μg of protein on SDS-PAGE and transfer to PVDF membranes .

• Antibody dilution: Use EYA4 antibody at dilutions ranging from 1:500 to 1:3000 based on antibody sensitivity .

• Incubation conditions: Incubate with primary antibody overnight at 4°C .

• Detection: For biotin-conjugated antibodies, use streptavidin-HRP conjugate for detection. For unconjugated antibodies, use appropriate HRP-conjugated secondary antibodies followed by enhanced chemiluminescence detection.

• Expected band size: Anticipate bands at approximately 67-70 kDa, consistent with the calculated molecular weight of 70 kDa for the 639 amino acid EYA4 protein .

For quantitative analysis, normalized band intensity can be measured using systems such as the FluorChem FC2 imaging system as described in published research .

How can biotin-conjugated EYA4 antibody be used to investigate EYA4's role in PLK1 regulation and mitotic processes?

Recent research has established that EYA4 promotes PLK1 activity through regulation of phosphorylation at the T210 site . To investigate this function using biotin-conjugated EYA4 antibody:

• Dual immunostaining approach: Combine the biotin-conjugated EYA4 antibody with antibodies against PLK1 phosphorylation targets (pS46 TCTP, pS198 CDC25C, pS133 Cyclin B) in mitotic cells.

• EYA4 knockdown studies: Compare immunostaining patterns of PLK1 substrates in control versus EYA4-depleted cells using siRNA or CRISPR-Cas9 systems.

• Co-immunoprecipitation: Use the biotin-conjugated antibody to pull down EYA4 complexes, followed by detection of associated PLK1 or its regulators.

• Functional rescue experiments: After EYA4 knockdown, reintroduce either wild-type EYA4 or phosphatase-inactive mutants (S128A, D375N) to assess restoration of PLK1 activity as measured by pT210 levels .

The experimental design should include appropriate controls to distinguish between direct and indirect effects on PLK1 regulation, as research has shown that EYA4 depletion significantly reduces phosphorylation of multiple PLK1 substrates in mitotic cells .

What approaches can be used to validate EYA4 antibody specificity in experimental systems?

Validating antibody specificity is crucial for obtaining reliable research results. For biotin-conjugated EYA4 antibody, consider the following validation approaches:

• Genetic knockdown controls: Perform siRNA or shRNA-mediated knockdown of EYA4 in experimental cell lines and confirm reduced signal in immunoassays, as demonstrated in published research where EYA4 knockdown efficiency was verified by Western blot .

• Xenograft validation: Generate xenograft tumors using cells with EYA4 knockdown and perform immunohistochemistry to verify reduced antibody staining, as described in published protocols .

• Multiple antibody comparison: Compare results from different antibodies targeting different epitopes of EYA4.

• Isotype control experiments: Include appropriate IgG isotype controls in all immunostaining experiments to assess background and non-specific binding .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to demonstrate specificity through signal reduction.

Successful validation increases confidence in experimental findings and should be considered an essential step in all EYA4-related research.

What are the common challenges when using biotin-conjugated antibodies in tissues with endogenous biotin?

When using biotin-conjugated EYA4 antibody in tissues with high endogenous biotin (such as liver, kidney, and brain), researchers may encounter false-positive signals. To address this issue:

• Endogenous biotin blocking: Pretreat tissue sections with avidin/biotin blocking kit before antibody application.

• Alternative detection systems: Consider using non-biotin amplification systems for tissues with high endogenous biotin.

• Positive and negative control tissues: Include tissues known to express high levels of EYA4 (heart, skeletal muscle) as positive controls and appropriate negative controls to establish staining specificity.

• Sample-specific optimization: As noted in the literature, antibody performance can be sample-dependent, so optimization may be required for each experimental system .

How can researchers accurately quantify EYA4 expression in immunohistochemistry experiments?

For rigorous quantification of EYA4 expression in immunohistochemical studies, two scoring systems have been validated in the literature:

System 1: Percentage-based semi-quantitative scoring

System 2: H-score method
The H-score is calculated using the formula: H-score = ΣPi (i + 1), where:

• i is the intensity score (range 0-3)

• Pi is the percentage of stained cells at each intensity (range 0-100%)

This produces a score ranging from 100 (100% negative cells) to 400 (100% strongly stained cells). The median H-score can be used as a cut-off to divide samples into high and low expression groups .

Both systems have been used successfully in EYA4 research, with the choice depending on the specific experimental question and required precision.

How can biotin-conjugated EYA4 antibody be used to investigate EYA4's role in tumor progression?

EYA4 has been implicated in tumor progression through its effects on cell proliferation. To investigate this role using biotin-conjugated EYA4 antibody:

• Comparative expression analysis: Perform immunohistochemistry on tumor versus normal tissue samples to quantify differences in EYA4 expression levels using the H-score method described above .

• Correlation with proliferation markers: Co-stain tissues with EYA4 antibody and proliferation markers such as Ki67 (ab15580, dilution 1:200) to establish relationships between EYA4 expression and proliferative capacity .

• Apoptosis assessment: Compare EYA4 expression with apoptotic markers such as cleaved Caspase 9 (ab52298, dilution 1:100) to evaluate potential relationships with cell survival .

• In vivo modeling: Use xenograft models with EYA4 overexpression or knockdown to assess tumor growth rates. Published research has demonstrated that tumors derived from EYA4-overexpressing cells grew more rapidly (mean tumor volume 0.69 ± 0.14 cm³) than control tumors (0.36 ± 0.09 cm³) .

These approaches can provide insights into how EYA4 contributes to tumor development and progression, potentially identifying new therapeutic targets.

What are the considerations for using EYA4 antibodies in studying its function as a tyrosine phosphatase?

EYA4 functions as a tyrosine phosphatase, specifically dephosphorylating 'Tyr-142' of histone H2AX . When studying this enzymatic function:

• Phosphatase-dead mutants: Compare results with wild-type EYA4 to those with phosphatase-inactive mutants (such as D375N) which have been shown to affect PLK1 activation .

• Substrate identification: Use biotin-conjugated EYA4 antibody in pull-down assays followed by mass spectrometry to identify novel substrates.

• Phosphorylation status monitoring: Combine EYA4 immunostaining with phospho-specific antibodies targeting known or potential substrates to correlate EYA4 expression with substrate phosphorylation status.

• Biochemical assays: Develop in vitro phosphatase assays using immunopurified EYA4 (via the biotin tag) and synthetic phosphopeptides representing potential substrates.

Understanding EYA4's phosphatase activity has significant implications for its role in various cellular processes, including DNA damage response and transcriptional regulation.

What are the optimal storage conditions for maintaining biotin-conjugated EYA4 antibody activity?

To maintain optimal activity of biotin-conjugated EYA4 antibody:

• Storage temperature: Store at -20°C for long-term preservation. The antibody remains stable for up to 12 months under these conditions .

• Formulation: The antibody is provided in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol .

• Aliquoting: For antibodies provided in larger volumes, aliquot upon receipt to avoid repeated freeze-thaw cycles, though the 20μl size formulations typically contain 0.1% BSA and may not require aliquoting for -20°C storage .

• Handling precautions: Note that the preservative ProClin is classified as poisonous and hazardous, requiring handling by trained staff only .

• Working solution: When preparing working dilutions, use freshly prepared buffers and store any remaining diluted antibody at 4°C for short-term use only.

Proper storage and handling are essential for maintaining antibody specificity and sensitivity over time, ensuring reproducible experimental results.