Cleaved-EFNA2 (N188) Antibody

What is the Cleaved-EFNA2 (N188) Antibody and what does it detect?

The Cleaved-EFNA2 (N188) Antibody is a rabbit polyclonal antibody that specifically detects endogenous levels of activated Ephrin-A2 protein fragments resulting from cleavage adjacent to the N188 position. This antibody binds to the amino acid region 110-190 in the C-terminal portion of human EFNA2. The specificity of this antibody is significant because it recognizes the cleaved form rather than just the full-length protein, enabling researchers to study post-translational modifications of Ephrin-A2 that may be relevant in various biological processes, particularly in cancer research .

What is the significance of cleaved Ephrin-A2 in biological research?

Cleaved forms of Ephrin-A2 represent an important area of study because they result from post-translational modifications that occur in specific biological contexts, including tumor microenvironments. Recent research has demonstrated that matrix metalloproteinases, particularly MT1-MMP, cleave EPHA2 (the receptor for EFNA2) around the N-terminal fibronectin type III (FnIII) domain, producing both soluble fragments and membrane-anchored truncated forms. Similar cleavage mechanisms likely affect EFNA2. These cleavage events may significantly alter signaling pathways and cellular functions, making the detection of cleaved forms particularly relevant for cancer research and understanding cell-cell communication mechanisms .

How does the antibody's binding specificity compare to other EFNA2 antibodies?

Unlike antibodies that target the full-length Ephrin-A2 protein, the Cleaved-EFNA2 (N188) Antibody specifically recognizes the fragment generated after cleavage at N188. This unique specificity allows researchers to distinguish between intact and processed forms of the protein in experimental samples. The antibody was generated using a synthesized peptide derived from human EFNA2 at the amino acid range 139-188 and was affinity-purified to ensure high specificity for this region. Western blot validation with etoposide-treated 293 cells confirms this specificity, as demonstrated by the blocking effect when the synthesized peptide is introduced .

What are the recommended applications and protocols for using the Cleaved-EFNA2 (N188) Antibody?

The Cleaved-EFNA2 (N188) Antibody has been validated for Western Blot (WB) and ELISA applications. For Western blot analysis, the recommended dilution range is 1:500-1:2000, while for ELISA, a higher dilution of 1:20000 is suggested. When performing Western blot, researchers should consider using etoposide-treated positive control cells (such as 293 cells treated with 25μM etoposide for 1 hour) to confirm antibody specificity. Inclusion of a blocking peptide control is also valuable for validation. The antibody may be suitable for other immunological techniques, but these applications require further validation by individual researchers .

How should samples be prepared to optimize detection of cleaved EFNA2?

To optimize detection of cleaved EFNA2 in experimental samples, researchers should consider treatments that induce cleavage events. Based on available data, etoposide treatment (25μM for 1 hour) has been shown to increase the detectable levels of cleaved EFNA2 in 293 cells. Additionally, considering that metalloproteinases like MT1-MMP are involved in the cleavage of related proteins, researchers may consider experimental conditions where these enzymes are active or overexpressed. For protein extraction, standard lysis buffers containing protease inhibitors are recommended to prevent further proteolytic degradation during sample preparation. Samples should be promptly processed and maintained at appropriate temperatures to preserve the cleaved fragments .

What are the essential experimental controls when working with this antibody?

When designing experiments using the Cleaved-EFNA2 (N188) Antibody, several controls are essential for result validation:

• Peptide Competition Assay: Including a lane where the antibody is pre-incubated with the synthesized immunogenic peptide to demonstrate binding specificity.

• Positive Control: Using cells known to express cleaved EFNA2, such as etoposide-treated 293 cells.

• Loading Control: Employing housekeeping protein antibodies to ensure equal loading across lanes.

• Cell Line Panel: Testing multiple cell lines with varying EFNA2 expression levels to confirm antibody sensitivity and specificity.

• Knockout/Knockdown Controls: If available, including EFNA2 knockout or knockdown samples to validate signal specificity.

These controls collectively ensure that signals detected truly represent cleaved EFNA2 rather than non-specific binding or technical artifacts .

How can the Cleaved-EFNA2 (N188) Antibody be utilized to study cancer progression mechanisms?

The Cleaved-EFNA2 (N188) Antibody offers valuable insights into cancer progression mechanisms through several experimental approaches. Researchers can investigate the correlation between cleaved EFNA2 levels and tumor aggressiveness by comparing clinical samples from different cancer stages. The antibody can be used to monitor changes in EFNA2 cleavage following treatment with therapeutic agents, potentially identifying molecular markers of treatment response. Additionally, researchers can explore the relationship between EFNA2 cleavage and the activity of matrix metalloproteinases in the tumor microenvironment. Based on research with the related EPHA2 receptor, where truncated forms play significant roles in tumor biology, similar investigations of EFNA2 cleavage may reveal important oncogenic mechanisms .

What is known about the relationship between EFNA2 cleavage and EphA receptor signaling?

The cleavage of EFNA2 likely impacts signaling through EphA receptors, although specific mechanisms are still being elucidated. Current research on the related EPHA2 receptor indicates that MT1-MMP cleaves EPHA2 at several sites in the N-terminal FnIII domain, producing membrane-anchored forms beginning with Y385, T395, V432, or N435. The anti-EPHA2 antibody DS-8895a was designed to recognize both full-length and these truncated forms. By analogy, EFNA2 cleavage may similarly modify ligand-receptor interactions in the Eph/ephrin signaling axis. Researchers using the Cleaved-EFNA2 (N188) Antibody can investigate whether cleaved forms of EFNA2 exhibit altered binding affinity to EphA receptors, potentially leading to differential downstream signaling events compared to the full-length ligand .

How might post-translational modifications affect antibody recognition of cleaved EFNA2?

Post-translational modifications (PTMs) can significantly impact antibody recognition of cleaved EFNA2. The Cleaved-EFNA2 (N188) Antibody targets the amino acid region 110-190, which may contain sites for phosphorylation, glycosylation, or other modifications. These PTMs could either enhance or hinder antibody binding, depending on whether they alter the epitope structure or accessibility. Researchers should consider potential phosphorylation sites within the target region, especially in experimental conditions where kinase activity is modulated. Additionally, glycosylation patterns may vary between different cell types or disease states, potentially affecting antibody recognition. When unexpected results occur, researchers should consider performing enzymatic deglycosylation or phosphatase treatments to evaluate the impact of these modifications on antibody binding .

What are common issues encountered when using the Cleaved-EFNA2 (N188) Antibody and how can they be addressed?

Researchers working with the Cleaved-EFNA2 (N188) Antibody may encounter several technical challenges:

• Weak Signal: This may result from low expression of cleaved EFNA2 in the sample. Consider using positive controls (etoposide-treated 293 cells), optimizing protein extraction methods, increasing antibody concentration, or extending exposure times.

• Multiple Bands: The presence of multiple bands could indicate different cleavage products, degradation, or non-specific binding. Verify specificity using peptide competition assays and optimize blocking conditions.

• High Background: This common problem may be addressed by increasing blocking time/concentration, optimizing antibody dilution, or adding additional washing steps.

• Inconsistent Results: Maintain consistent experimental conditions, including sample preparation, antibody dilution, and incubation times. Aliquot the antibody to avoid repeated freeze-thaw cycles.

• Loss of Activity: Store the antibody at -20°C in small aliquots to prevent freeze-thaw degradation, as recommended by the manufacturer .

What quality control metrics should be established when implementing this antibody in a new research project?

When implementing the Cleaved-EFNA2 (N188) Antibody in a new research project, establishing robust quality control metrics is essential:

• Reproducibility Assessment: Perform replicate experiments to determine the coefficient of variation for key measurements.

• Sensitivity Determination: Establish detection limits using dilution series of positive control samples.

• Specificity Validation: Confirm signal specificity through peptide competition assays and, if possible, genetic knockdown/knockout controls.

• Cross-Reactivity Testing: Evaluate potential cross-reactivity with other ephrin family members, particularly those with sequence homology in the target region.

• Lot-to-Lot Consistency: Compare results between different antibody lots when replacing depleted stock.

• Storage Stability Analysis: Monitor antibody performance over time to establish optimal storage conditions and usage timeframes.

These quality control procedures should be documented and standardized to ensure consistent, reliable results throughout the research project .

How can researchers validate that the detected signals truly represent cleaved EFNA2?

Validating that detected signals represent cleaved EFNA2 requires multiple complementary approaches:

• Peptide Competition: Pre-incubating the antibody with the immunogenic peptide should abolish or significantly reduce specific signals.

• Size Verification: The cleaved fragment should appear at the expected molecular weight (~15-20 kDa, depending on the precise cleavage site).

• Induction Experiments: Treatments known to induce EFNA2 cleavage (e.g., etoposide) should increase signal intensity.

• Protease Inhibition: Including specific metalloproteinase inhibitors during cell culture should reduce the levels of cleaved EFNA2 if MT1-MMP or similar enzymes are responsible for the cleavage.

• Mass Spectrometry: For definitive validation, cleaved fragments can be immunoprecipitated and identified using mass spectrometry to confirm the cleavage site.

• Orthogonal Detection Methods: Using alternative detection methods or antibodies targeting different regions of EFNA2 to corroborate findings .

What are the emerging research directions for cleaved EFNA2 in biomedical research?

Emerging research directions for cleaved EFNA2 in biomedical research span several promising areas. The role of cleaved EFNA2 in cancer biology merits further investigation, particularly regarding its potential contribution to tumor invasion and metastasis. Researchers might explore how EFNA2 cleavage alters the tumor microenvironment and influences interactions between cancer cells and stromal components. Additionally, the potential utility of cleaved EFNA2 as a biomarker for cancer progression or treatment response represents an exciting translational direction. Drawing parallels with EPHA2 research, where the afucosylated antibody DS-8895a has shown therapeutic potential, similar approaches targeting cleaved EFNA2 might yield novel therapeutic strategies. Finally, exploring the relationship between EFNA2 cleavage and other post-translational modifications could reveal sophisticated regulatory mechanisms in normal physiology and disease states .