EN2 Antibody

What is EN2 protein and what are its structural characteristics?

EN2 (engrailed homeobox 2) is a homeodomain-containing transcription factor with a canonical length of 333 amino acid residues and a molecular mass of 34.2 kDa in humans. It belongs to the Engrailed homeobox protein family and is primarily localized in the nucleus. The protein contains three alpha helices: helices 1 and 2 at the N-terminus bind DNA, while helix 3 at the C-terminus mediates exocrine and internalization functions of EN2 . EN2 plays essential roles in neuronal differentiation and regulation of apoptosis. Its gene orthologs have been identified in multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken .

What applications are commonly used with EN2 antibodies?

EN2 antibodies are utilized in multiple experimental applications:

These applications enable researchers to detect, quantify, and localize EN2 in various experimental settings .

How can I determine the specificity of my EN2 antibody?

Determining antibody specificity is critical due to documented inconsistencies with some commercial EN2 antibodies. A comprehensive validation approach should include:

• Western blot analysis using both endogenous and exogenous (overexpressed) EN2 protein. Compare lysates from cells known to express EN2 with those that do not or with EN2-knockdown cells.

• Immunofluorescence studies to determine subcellular localization patterns. True EN2 protein typically shows nuclear localization for endogenous protein, though exogenous overexpressed EN2 has been observed in both nuclear and cytoplasmic compartments .

• Testing with negative controls such as isotype control antibodies and pre-absorption with the immunizing peptide.

• Cross-validation using multiple antibodies targeting different epitopes of EN2. One study demonstrated that anti-EN2 antibody ab28731 and anti-EN2 antibody MAB2600 showed discrepant staining patterns, with the former showing cytoplasmic localization and the latter showing nuclear localization .

How should I optimize immunohistochemical staining with EN2 antibodies?

For optimal immunohistochemical detection of EN2:

• Antibody concentration: 5 μg/mL has been determined as optimal in some studies .

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is commonly effective.

• Validation controls: Include positive controls (tissues known to express EN2) and negative controls (antibody diluent only).

• Consider both staining intensity and pattern: Research has revealed that EN2 may show differential localization in normal versus cancerous tissues. For example, in benign prostatic hyperplasia (BPH), EN2 staining appears primarily in the nucleus and cytoplasm, while in prostate cancer (PC), staining is predominantly on the cytomembrane .

• Scoring system: Develop a quantitative scoring system for both staining intensity and percentage of positive cells to enable objective comparison between samples.

How do EN2 expression patterns differ between normal and cancerous tissues?

Research has demonstrated distinct differences in EN2 expression between normal and cancerous tissues:

• Prostate cancer vs. BPH: Immunohistochemical staining shows significantly stronger signals in PC compared to BPH, indicating higher EN2 expression levels in PC. This finding has been confirmed by RT-PCR analysis .

• Subcellular localization differences: EN2 staining in BPH tissues is primarily nuclear and cytoplasmic, while in PC tissues, it is predominantly observed on the cytomembrane .

• Expression correlation with disease progression: Studies have found that EN2 expression levels positively correlate with PC clinical staging, suggesting its potential value as a prognostic marker .

• Renal cell carcinoma: Interestingly, a protein detected by one anti-EN2 antibody (termed "nonEN2") showed lower expression in kidney tumor tissues compared to normal kidney tissues, contrary to the pattern observed in prostate cancer .

What novel detection methods have been developed for EN2 protein?

Recent advances include:

• Hybridization Chain Reaction (HCR)-based aptamer-antibody hybrid Enzyme-Linked Oligonucleotide Assay (ELONA): This system combines the specificity of antibody-antigen interactions with the signal amplification capabilities of nucleic acid hybridization chain reactions. It uses an EN2-specific aptamer (Kd = 8.26 nM) connected to a trigger sequence that initiates HCR, resulting in formation of DNA nanostructures that amplify detection signals .

• Performance metrics of HCR-ELONA:

  • Detection time: <2.5 hours

  • Limit of detection: 0.34 nM in buffer and 2.69 nM in artificial urine

  • High specificity: Does not detect other urinary proteins

  • Stability: Maintains performance at 4°C for at least three months

• This method has potential applications in non-invasive detection of bladder and prostate cancers using urine samples, as EN2 has been detected at elevated levels in the urine of PC patients .

Why do different EN2 antibodies show discrepant staining patterns?

The discrepancy in staining patterns between different EN2 antibodies has been documented and may be attributed to:

• Epitope specificity: Different antibodies target different regions of the EN2 protein. For example, one study developed a monoclonal antibody specifically targeting Helix 3 of EN2, which is involved in exocrine and internalization functions .

• Antibody specificity issues: Research has revealed that some commercial antibodies may not bind to the intended target. One study found that anti-EN2 antibody ab28731 did not react with lysates from cells overexpressing full-length EN2, suggesting it may be detecting a novel protein that was termed "nonEN2" .

• Post-translational modifications: Different antibodies may have differential sensitivity to post-translational modifications of EN2.

• Tissue-specific expression patterns: EN2 may undergo different processing or have different interacting partners in various tissues, affecting epitope availability.

When encountering inconsistent results, researchers should validate their antibodies using multiple techniques and consider using antibodies targeting different epitopes for confirmation .

How can EN2 antibodies be used to investigate the role of EN2 in cancer progression?

EN2 has been identified as a candidate oncogene in several cancers. Here are methodological approaches using EN2 antibodies:

• Correlation studies: Use immunohistochemistry with validated EN2 antibodies to score EN2 expression in patient samples across different cancer stages, then correlate with clinical parameters to assess prognostic value .

• Functional studies: Combine EN2 immunodetection with proliferation, migration, or invasion assays to investigate its role in cancer cell behavior.

• Biomarker development: Employ ELISA or novel detection methods like HCR-ELONA to quantify EN2 in body fluids (e.g., urine) for potential non-invasive diagnostic applications .

• Subcellular localization studies: Use immunofluorescence to track changes in EN2 localization during disease progression, as shifts from nuclear to cytoplasmic or membrane localization may indicate functional changes .

• Targeted therapy development: EN2 has been evaluated as a potential target for vaccine strategies in ovarian cancer, where HLA-A2 restricted epitopes from the EN2 protein have been identified using computer algorithms and tested for immunogenicity .

What emerging applications are being developed for EN2 antibodies?

Several promising research directions include:

• Development of targeted immunotherapies: Studies are exploring EN2 as a target for vaccine strategies in multiple cancers, including ovarian cancer. Researchers have identified potentially immunogenic HLA-A2 restricted epitopes from the EN2 protein and tested T cell responses against these peptides .

• Non-invasive diagnostic tools: The development of sensitive detection methods like aptamer-antibody hybrid assays for EN2 in urine samples could lead to non-invasive early detection tests for prostate and bladder cancers .

• Combination diagnostics: Integrating EN2 detection with other biomarkers may improve diagnostic accuracy and provide more comprehensive disease profiling.

• Therapeutic antibodies: Given EN2's potential role in cancer progression, therapeutic antibodies targeting EN2 could be developed, particularly for cancers with membrane localization of EN2.

• Circulating tumor cell detection: EN2 antibodies might be utilized in the identification and characterization of circulating tumor cells, aiding in monitoring disease progression and treatment response.