EFL4 Antibody

What is EFL4 and how does it relate to EFNA4?

EFL4 (Eph-Related Receptor Tyrosine Kinase Ligand 4) is an alternative name for EFNA4 (Ephrin A4). Other alternative names include LERK-4, EPLG4, and Ligand of Eph-Related Kinase 4. EFNA4 is a protein involved in the EPH-Ephrin signaling pathway, which plays crucial roles in nervous system development and is associated with conditions such as Craniofrontonasal Syndrome and Craniosynostosis . This protein is encoded by gene ID 1945 and its UniProt accession number is P52798, making it a well-characterized component of developmental signaling systems.

What are the key characteristics of recombinant anti-EFNA4 antibodies?

Recombinant anti-EFNA4 antibodies, such as the humanized monoclonal antibody V3S-0324-FY71, demonstrate specific binding affinity to human ephrin-A4 with a KD value of 4.8 nM . These antibodies typically exhibit cross-reactivity with ephrin-A4 from other species, including mouse and cynomolgus ephrin-A4 . The high specificity and defined affinity make these antibodies valuable tools for detecting and studying ephrin-A4 in multiple experimental systems. Production in mammalian expression systems (HEK293F or CHO cells) ensures proper folding and post-translational modifications critical for epitope recognition.

How are anti-EFNA4 antibodies classified and what implications does this have for research?

Anti-EFNA4 antibodies are typically classified based on multiple parameters including isotype (e.g., human IgG1), clonality (monoclonal vs. polyclonal), species reactivity, and epitope specificity . This classification impacts their application suitability - monoclonal antibodies offer consistent specificity across experiments but may recognize only a single epitope, while polyclonal preparations provide broader epitope recognition but potentially greater batch-to-batch variation. The isotype affects downstream detection methods and potential functional activities, such as complement activation or Fc receptor binding, which may be relevant in certain experimental contexts.

What experimental protocols maximize the utility of anti-EFNA4 antibodies in ELISA applications?

For optimal ELISA performance with anti-EFNA4 antibodies, the following methodological approach is recommended:

• Coating concentration: Use purified recombinant EFNA4 at 1-2 μg/ml in carbonate buffer (pH 9.6)

• Blocking: 2-3% BSA or 5% non-fat milk in PBS for 1-2 hours at room temperature

• Primary antibody: Apply anti-EFNA4 antibody at 0.5 μg/ml in blocking buffer

• Detection system: HRP-conjugated secondary antibody specific to the primary antibody isotype

• Substrate: TMB solution followed by stop solution (2N H₂SO₄)

• Controls: Include both positive controls (known EFNA4 samples) and negative controls (buffer only and irrelevant proteins)

This protocol should be optimized for each specific antibody and sample type. Including a standard curve with recombinant EFNA4 protein allows for quantitative analysis, while careful attention to washing steps (typically 3-5 washes with PBS-T between each step) minimizes background signal and improves sensitivity.

How should researchers approach Western blot optimization when using anti-EFNA4 antibodies?

Western blot optimization for EFNA4 detection requires careful consideration of multiple parameters:

• Sample preparation: Consider both denaturing and non-denaturing conditions, as some epitopes may be conformation-dependent

• Protein loading: 20-50 μg of total protein per lane for cell/tissue lysates

• Gel percentage: 10-12% SDS-PAGE gels typically provide good resolution for EFNA4 (predicted MW ~28 kDa)

• Transfer conditions: Semi-dry or wet transfer at 25V overnight at 4°C for efficient protein transfer

• Blocking: 5% non-fat milk or 3-5% BSA in TBS-T (the latter preferred if using phospho-specific antibodies)

• Primary antibody incubation: Dilute according to manufacturer recommendations, typically in blocking buffer overnight at 4°C

• Validation: Use positive controls (tissues known to express EFNA4) and negative controls (knockdown samples)

Researchers should validate band specificity through peptide competition assays, where pre-incubation of the antibody with the immunizing peptide should abolish specific bands if the antibody is truly specific for EFNA4.

What are the recommended storage and handling protocols for maintaining anti-EFNA4 antibody performance?

To preserve antibody function and extend shelf-life, the following storage and handling recommendations should be implemented:

Researchers should avoid repeated freeze-thaw cycles, which can cause antibody denaturation and aggregation leading to reduced binding affinity and increased non-specific binding. Working aliquots should be prepared during initial thawing to minimize the need to thaw the stock solution repeatedly.

How can anti-EFNA4 antibodies be utilized to investigate EPH-Ephrin signaling mechanisms in neural development?

Investigation of EPH-Ephrin signaling using anti-EFNA4 antibodies requires sophisticated experimental approaches:

• Functional blocking studies: Using anti-EFNA4 antibodies to interrupt receptor-ligand interactions and observe phenotypic consequences in neuronal cultures or developmental models

• Immunohistochemistry of developmental time series: Mapping the spatiotemporal expression of EFNA4 across neural development stages using validated antibodies

• Co-immunoprecipitation experiments: Pulling down EFNA4 along with binding partners to map protein interaction networks

• Proximity ligation assays: Detecting in situ interactions between EFNA4 and EphA receptors with single-molecule sensitivity

• Combined genetic and antibody approaches: Using CRISPR-edited cells with specific EFNA4 mutations followed by antibody detection to correlate structure with function

These methodological approaches can help elucidate the role of EFNA4 in establishing neuronal connectivity, axon guidance, and tissue boundary formation, relevant to understanding both normal development and pathological conditions like Craniofrontonasal Syndrome .

What strategies should be employed when validating anti-EFNA4 antibody specificity for critical research applications?

Comprehensive validation of anti-EFNA4 antibodies should employ multiple orthogonal approaches:

• Genetic validation: Test antibody reactivity in EFNA4 knockout/knockdown models versus wild-type controls

• Epitope mapping: Determine the exact binding region to assess potential cross-reactivity with other ephrin family members

• Multi-application consistency: Verify that the antibody recognizes EFNA4 across different techniques (WB, IHC, IP, ELISA)

• Mass spectrometry correlation: Confirm that immunoprecipitated proteins detected by the antibody are identified as EFNA4 by MS

• Comparative analysis: Test multiple anti-EFNA4 antibodies targeting different epitopes to build confidence in detection

• Cross-species reactivity testing: Evaluate performance across human, mouse, and other relevant species as claimed by manufacturers

This systematic validation approach is particularly important for EFNA4 research due to the high sequence homology among ephrin family members and the existence of multiple isoforms that may be differentially recognized by antibodies.

How do epitope differences among anti-EFNA4 antibodies affect functional studies in EPH-Ephrin signaling research?

The specific epitope recognized by an anti-EFNA4 antibody can profoundly impact experimental outcomes in signaling studies:

• Receptor-binding domain epitopes: Antibodies targeting regions involved in receptor binding may block signaling, making them useful for inhibition studies but potentially confounding for detection in functional contexts

• Membrane-association domain epitopes: Antibodies recognizing the GPI-anchor processing region may differentially detect membrane-bound versus soluble forms of EFNA4

• Post-translational modification sites: Some antibodies may specifically recognize or be blocked by phosphorylation, glycosylation, or other modifications of EFNA4

• Conformation-dependent epitopes: Certain antibodies may only recognize EFNA4 in its native conformation but not in denatured states

• Clustering-sensitive recognition: Some antibodies may have altered affinity for EFNA4 when it is clustered, which is physiologically relevant as ephrin clustering is important for receptor activation

Understanding these epitope-related factors is critical when designing experiments to study EFNA4's role in bidirectional signaling, cell adhesion, and receptor clustering - all key aspects of EPH-Ephrin biology.

What approaches can resolve contradictory results when using different anti-EFNA4 antibodies in the same experimental system?

When faced with discrepant results using different antibodies, researchers should implement a systematic resolution strategy:

• Epitope mapping: Determine if the antibodies recognize different regions of EFNA4 that may be differentially accessible in particular experimental conditions

• Application-specific validation: Re-validate each antibody specifically for the application where discrepancies arose

• Isoform analysis: Investigate whether differences reflect detection of distinct EFNA4 isoforms by performing RT-PCR to identify which isoforms are expressed in the system

• Cross-reactivity assessment: Test each antibody against related ephrin family members to identify potential off-target binding

• Orthogonal methods: Employ non-antibody-based detection methods (e.g., RNA-seq, mass spectrometry) to resolve contradictions

• Functional validation: Use genetic approaches (siRNA, CRISPR) to manipulate EFNA4 expression and correlate with antibody detection patterns

This methodical approach can help distinguish genuine biological insights from technical artifacts, particularly important given the complex regulation and multiple functions of ephrin family proteins.

How can researchers optimize immunohistochemistry protocols for detecting native EFNA4 in neural tissues?

Optimizing immunohistochemistry for EFNA4 detection in neural tissues requires addressing several tissue-specific challenges:

• Fixation optimization: Test multiple fixation methods (4% PFA, methanol, Bouin's) as ephrin epitopes can be fixation-sensitive

• Antigen retrieval: Compare heat-induced (citrate buffer, pH 6.0) versus enzymatic retrieval methods to maximize epitope accessibility

• Permeabilization: Gentle detergent treatment (0.1-0.3% Triton X-100) balanced to maintain tissue integrity while allowing antibody access

• Signal amplification: Consider tyramide signal amplification for low-abundance detection while maintaining specificity

• Autofluorescence reduction: Implement sodium borohydride treatment or Sudan Black B to reduce lipofuscin autofluorescence common in neural tissues

• Co-localization controls: Include markers for specific neural cell types to contextualize EFNA4 expression patterns

• Z-stack acquisition: Collect optical sections to generate 3D reconstructions of EFNA4 distribution in complex neural structures

These optimizations help overcome the inherent challenges of detecting potentially low-abundance signaling molecules like EFNA4 in heterogeneous neural tissue environments.

What methodological approaches can distinguish between different functional states of EFNA4 using antibody-based detection?

Advanced techniques for discriminating EFNA4 functional states include:

• Phospho-specific antibodies: Development or selection of antibodies that specifically recognize phosphorylated forms of EFNA4 associated with activation

• Conformation-sensitive antibodies: Utilizing antibodies that selectively bind active versus inactive conformations of EFNA4

• Proximity-based assays: Implementing FRET or BRET approaches to detect EFNA4-receptor interactions in real-time

• Single-molecule imaging: Applying super-resolution techniques to visualize EFNA4 clustering during activation

• Antibody-based biosensors: Developing FRET-based sensors using anti-EFNA4 antibody fragments to detect conformational changes

• Sequential immunoprecipitation: Using different antibodies recognizing distinct EFNA4 epitopes to separate subpopulations

• Native versus denatured detection comparison: Parallel analysis under conditions that maintain or disrupt protein conformation

These sophisticated approaches permit researchers to move beyond simple detection of EFNA4 presence toward understanding its dynamic functional states in complex biological systems.

How are anti-EFNA4 antibodies being adapted for multiplexed imaging approaches in neurodevelopmental research?

Cutting-edge multiplexed imaging with anti-EFNA4 antibodies employs several advanced methodologies:

• Cyclic immunofluorescence: Sequential staining/imaging/quenching cycles allowing detection of dozens of proteins including EFNA4 and its signaling partners

• Mass cytometry imaging: Metal-conjugated anti-EFNA4 antibodies enabling simultaneous detection of 40+ proteins on tissue sections

• DNA-barcoded antibodies: Anti-EFNA4 antibodies linked to oligonucleotide barcodes for highly multiplexed CODEX imaging

• Quantum dot multiplexing: Conjugating anti-EFNA4 antibodies to quantum dots with discrete emission spectra for spectral unmixing

• Spatial transcriptomics integration: Combining anti-EFNA4 immunodetection with in situ RNA sequencing to correlate protein and transcript distributions

These approaches facilitate comprehensive mapping of EFNA4 expression patterns relative to other signaling components across developmental timepoints, enabling systems-level understanding of EPH-Ephrin network function in neurodevelopment.

What considerations are important when developing anti-EFNA4 antibodies for potential therapeutic applications in neurodevelopmental disorders?

Development of therapeutic anti-EFNA4 antibodies requires addressing several critical factors:

• Epitope selection: Targeting epitopes that specifically modulate disease-relevant interactions rather than all EFNA4 functions

• Blood-brain barrier penetration: Engineering antibody formats (e.g., bispecific antibodies with transferrin receptor targeting) to facilitate CNS delivery

• Developmental timing: Designing administration regimens that target specific developmental windows where EFNA4 modulation would be beneficial

• Functional selectivity: Creating antibodies that selectively inhibit or enhance specific EFNA4 interactions with particular EphA receptors

• Safety profiling: Thoroughly characterizing potential off-target effects given EFNA4's roles in multiple developmental processes

• Humanization considerations: Ensuring therapeutic antibodies are fully humanized to minimize immunogenicity in clinical applications

• Dosing strategies: Determining optimal concentration ranges that modulate pathological signaling without disrupting physiological functions

While primarily research-focused currently, these considerations highlight the potential translation of basic EFNA4 antibody research toward therapeutic applications for neurodevelopmental disorders associated with EPH-Ephrin signaling dysregulation.