EPHA8 Antibody, Biotin conjugated

What is EPHA8 and why is it an important research target?

EPHA8 (Ephrin type-A receptor 8) is a receptor tyrosine kinase belonging to the Eph family, which constitutes the largest family of tyrosine kinase receptors in the human genome. EPHA8 functions as a receptor that binds promiscuously to GPI-anchored ephrin-A family ligands (particularly EFNA2, EFNA3, and EFNA5) on adjacent cells, leading to contact-dependent bidirectional signaling .

Its significance stems from its roles in:

• Regulating integrin-mediated cell adhesion and migration on fibronectin substrate

• Promoting neurite outgrowth during neural development

• Axon guidance in the developing nervous system

• Participating in cell-cell communication via both forward signaling (downstream of the receptor) and reverse signaling (downstream of the ephrin ligand)

Notably, EPHA8 can function through both kinase-dependent and kinase-independent mechanisms, making it an intriguing target for understanding diverse cellular signaling pathways .

How does the biotin conjugation process affect EPHA8 antibody functionality?

Biotin conjugation to EPHA8 antibodies introduces measurable effects on antibody functionality that researchers must account for in experimental design:

When working with biotin-conjugated EPHA8 antibodies, researchers should validate each lot by comparing binding activity at different conjugation ratios to optimize experimental conditions.

What methods can be used to verify EPHA8 antibody specificity across different experimental systems?

Verifying antibody specificity is crucial for experimental validity. For EPHA8 antibodies, employ these complementary approaches:

• Cross-reactivity assessment against other Eph family members:

  • Conduct plate-based ELISA using recombinant EphA family members (EphA1-EphA8)

  • Proper specific antibodies should bind exclusively to EPHA8, not to other isoforms despite their structural similarities

• Flow cytometry validation:

  • Compare fluorescence intensity between cells expressing EPHA8 (e.g., BT-549 cells with huEphA8) versus mock controls

  • Quantify statistical significance of binding compared to isotype controls

• Knockout/knockdown controls:

  • Use CRISPR/Cas9-mediated EPHA8 knockout cells or siRNA knockdown models

  • Compare antibody binding between wild-type and knockout/knockdown samples

• Immunoprecipitation followed by mass spectrometry:

  • Capture proteins using the EPHA8 antibody

  • Analyze precipitated proteins by mass spectrometry to confirm EPHA8 identity

  • Examine for presence of known EPHA8-interacting partners (e.g., p110γ PI 3-kinase)

• Competitive binding assays:

  • Pre-incubate with free recombinant EPHA8 to block specific binding sites

  • A specific antibody will show significantly reduced signal following this competition

• Immunoblotting for size verification:

  • Confirm detection of a band at the expected molecular weight (111 kDa)

  • Compare band patterns between tissues known to express versus not express EPHA8

How can biotin-conjugated EPHA8 antibodies be optimally used in co-immunoprecipitation experiments?

For successful co-immunoprecipitation (Co-IP) experiments with biotin-conjugated EPHA8 antibodies:

• Sample preparation optimization:

  • Lyse cells in non-denaturing buffers (e.g., RIPA or NP-40 buffer with protease/phosphatase inhibitors)

  • For EPHA8 membrane protein complexes, include 1% digitonin or 0.5% CHAPS to preserve protein-protein interactions

• Strategic conjugation approach:

  • Use antibodies with optimal biotin:antibody ratio (typically 4-8 biotin per antibody)

  • Higher conjugation ratios may compromise binding affinity while too few biotin molecules limit detection sensitivity

• Capture method selection:

  • Utilize streptavidin-coated magnetic beads for efficient capture

  • Pre-clear lysates with uncoated beads to reduce non-specific binding

  • For studying EPHA8 activation, consider using preclustered ephrin A5-Fc proteins to stimulate cells prior to lysis

• Detection strategy:

  • Avoid using biotin-based detection methods that would interfere with the conjugated antibody

  • Use HRP-conjugated antibodies against expected interacting partners (e.g., anti-p110γ PI 3-kinase)

  • Consider using a non-biotin conjugated EPHA8 antibody recognizing a different epitope for verification

• Controls implementation:

  • Include IgG isotype control to assess non-specific binding

  • Use lysates from cells without EPHA8 expression as negative controls

  • For kinase activity studies, include samples treated with PI 3-kinase inhibitor wortmannin

• Validation approach:

  • Confirm co-precipitation using reciprocal IP (i.e., IP with partner protein antibody, detect EPHA8)

  • Analyze co-IP samples by western blot probing for known EPHA8 interacting proteins

What experimental approaches can differentiate between kinase-dependent and kinase-independent functions of EPHA8?

Distinguishing kinase-dependent from kinase-independent EPHA8 functions requires careful experimental design:

• Kinase-inactive EPHA8 mutant expression:

  • Generate expression constructs for wild-type EPHA8 and kinase-inactive mutants (e.g., K666M or K666R mutations in the ATP-binding site)

  • Express these in cell lines like NIH 3T3 or HEK293

  • Compare cellular phenotypes between wild-type and kinase-inactive conditions

• Phosphorylation status analysis:

  • Use phospho-specific antibodies against EPHA8 tyrosine residues

  • Perform western blotting to verify phosphorylation state differences between wild-type and kinase-inactive forms

  • For precise quantification, combine with phosphopeptide mass spectrometry

• Domain-specific mutants utilization:

  • Generate and express EPHA8 mutants with specific domain deletions:

    • Juxtamembrane segment deletion (EPHA8-ΔJM)

    • Sterile alpha motif deletion (EPHA8-ΔSAM)

  • Compare functions to determine domain requirements for specific activities

• Integrin-mediated adhesion assays:

  • Measure cell attachment to fibronectin in cells expressing wild-type versus kinase-inactive EPHA8

  • Research shows both forms enhance adhesion via α5β1 or β3 integrins, indicating kinase-independence of this function

• PI 3-kinase signaling pathway investigation:

  • Treat cells with wortmannin (PI 3-kinase inhibitor) to block EPHA8-promoted adhesion

  • Assess association between EPHA8 and p110γ isoform of PI 3-kinase

  • Use lipid kinase-inactive p110γ mutants to suppress EPHA8-promoted cell adhesion

• Ephrin ligand stimulation:

  • Stimulate cells with preclustered ephrin-A5-Fc proteins

  • Compare signaling responses between wild-type and kinase-inactive EPHA8

  • Examine cytoskeletal modifications using rhodamine-phalloidin staining

How does EPHA8 regulate integrin activity through p110γ PI 3-kinase, and how can researchers study this interaction?

EPHA8 regulation of integrin activity through p110γ PI 3-kinase represents a key kinase-independent pathway that can be investigated through several approaches:

• Biochemical interaction characterization:

  • Perform co-immunoprecipitation experiments using biotin-conjugated EPHA8 antibodies

  • Detect associated p110γ by western blotting

  • The juxtamembrane segment of EPHA8 is sufficient for forming a stable complex with p110γ

• Functional analysis protocol:

  • Express wild-type EPHA8 or the juxtamembrane deletion mutant (EPHA8-ΔJM)

  • Measure cell attachment to fibronectin substrate

  • The EPHA8-ΔJM mutant fails to enhance cell adhesion, confirming the critical role of this domain

• PI 3-kinase inhibition studies:

  • Treat cells with wortmannin at different concentrations (100-500 nM)

  • Evaluate dose-dependent inhibition of EPHA8-promoted adhesion

  • This approach establishes the dependence of integrin activation on PI 3-kinase activity

• Dominant-negative approach:

  • Express membrane-targeted lipid kinase-inactive p110γ mutant

  • This mutant stably associates with EPHA8 but suppresses EPHA8-promoted cell adhesion

  • Measurement of cell attachment to fibronectin quantifies this suppression

• Ligand stimulation experiments:

  • Remove endogenous ephrin A ligands by treating cells with PI-PLC

  • Stimulate with preclustered ephrin A5-Fc proteins

  • Assess integrin activation through cell spreading/adhesion assays

• Integrin activation measurement:

  • Use antibodies recognizing activated integrin conformations (e.g., HUTS-21 for activated β1)

  • Perform flow cytometry to quantify integrin activation levels

  • Compare between controls and cells expressing EPHA8 variants

What controls are essential when using biotin-conjugated EPHA8 antibodies in immunofluorescence studies?

Proper controls are critical for immunofluorescence studies using biotin-conjugated EPHA8 antibodies:

• Specificity controls:

  • Negative control tissues/cells known to lack EPHA8 expression

  • Competitive inhibition using recombinant EPHA8 protein to block specific binding

  • Secondary antibody-only control to assess background

• Signal validation approaches:

  • Compare staining pattern with a second non-biotin conjugated EPHA8 antibody targeting a different epitope

  • Use EPHA8 knockout or knockdown samples as negative controls

  • Validate subcellular localization by co-staining with established markers for relevant cellular compartments

• Biotin-specific considerations:

  • Block endogenous biotin using avidin/biotin blocking kits before applying biotin-conjugated antibodies

  • Include controls for potential streptavidin binding to endogenous biotinylated proteins

  • Use titratable detection approaches to optimize streptavidin-fluorophore concentration

• Technical controls:

  • Use an isotype-matched biotin-conjugated irrelevant antibody as negative control

  • Include positive controls with known EPHA8 expression patterns

  • Test for streptavidin detection system non-specific binding

• Cross-reactivity assessment:

  • Stain tissues from EPHA8 knockout mice to confirm absence of cross-reactivity with other Eph family members

  • Test antibody on cells expressing individual EphA family members to ensure specificity

How can researchers accurately quantify the effect of biotin conjugation load on EPHA8 antibody stability and function?

To systematically assess the impact of biotin conjugation load on EPHA8 antibody properties:

• Preparation of defined conjugation series:

  • Generate EPHA8 antibodies with varying biotin:antibody ratios (e.g., 2:1, 5:1, 10:1, 20:1, >50:1)

  • Precisely determine actual conjugation ratios using HABA assay or mass spectrometry

• Thermostability measurement:

  • Perform differential scanning calorimetry (DSC) across the conjugation series

  • Plot melting temperature (Tm) against biotin load to establish correlation

  • Different IgG scaffolds show varying sensitivity to conjugation, so this must be determined empirically

• Binding activity quantification:

  • Conduct ELISA using plate-bound recombinant EPHA8 protein

  • Measure binding activity across the conjugation series at equal antibody concentrations

  • Determine EC50 values to quantify changes in binding affinity

• Functional activity assessment:

  • For each conjugation ratio, compare:

    • Direct antigen binding (by ELISA)

    • Signal generation capability (via streptavidin-HRP binding)

  • Plot the relationship between conjugation level, binding activity, and signal strength

• Storage stability analysis:

  • Evaluate activity retention after storage at different temperatures (-80°C, -20°C, 4°C)

  • Monitor stability over time (1 week, 1 month, 3 months)

  • Identify optimal storage conditions for each conjugation ratio

• Fc receptor binding assessment:

  • Use surface plasmon resonance to measure binding to FcγRIIb (CD32b)

  • Determine if high biotin loads impact Fc receptor interactions, which could affect downstream applications

What methodological approaches can detect potential heterooligomerization between EPHA8 and other Eph receptors?

Investigating EPHA8's interaction with other Eph receptors requires specialized techniques:

• Co-immunoprecipitation with differential tagging:

  • Express EPHA8 with one tag (e.g., HA) and other Eph receptors with different tags (e.g., FLAG)

  • Perform IP with anti-HA and blot for FLAG to detect heterooligomerization

  • The reverse experiment (IP with anti-FLAG, blot for HA) should confirm the interaction

• Proximity ligation assay (PLA):

  • Use specific antibodies against EPHA8 and potential partner Eph receptors

  • PLA signal indicates close proximity (<40 nm) suggesting heterooligomerization

  • Quantify PLA dots per cell to measure interaction frequency

• Receptor biotinylation and selective recovery:

  • Engineer Eph receptors containing N-terminal biotin acceptor peptide (AP) tags

  • Express with E. coli biotin ligase (BirA) for selective biotinylation

  • Use streptavidin-coated magnetic Dynabeads to cluster and recover complexes

  • Western blot with α-EPHA8 antibodies to detect association with other biotinylated Eph receptors

• Förster resonance energy transfer (FRET):

  • Create fusion proteins of EPHA8-CFP and other Eph receptors-YFP

  • Measure FRET efficiency to quantify protein-protein interactions

  • Live-cell imaging allows visualization of interactions in real-time

• Bimolecular fluorescence complementation (BiFC):

  • Fuse EPHA8 with one half of a fluorescent protein (e.g., YFP-N)

  • Fuse other Eph receptors with complementary half (e.g., YFP-C)

  • Fluorescence occurs only when proteins interact to reconstitute the fluorophore

• Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS):

  • Analyze purified receptor ectodomains to determine oligomeric states

  • Compare size distributions of individual receptors versus mixtures

  • Shifts in molecular weight indicate complex formation

• Single-molecule imaging techniques:

  • Use total internal reflection fluorescence (TIRF) microscopy with differentially labeled receptors

  • Track co-diffusion and co-localization at the single-molecule level

  • Quantify interaction dynamics and duration

How can EPHA8 antibodies be used to investigate developmental changes in axonal projection patterns?

For studying EPHA8's role in axonal development:

• Developmental timing analysis:

  • Perform immunohistochemistry with biotin-conjugated EPHA8 antibodies at different embryonic stages

  • Correlate EPHA8 expression with key developmental events

  • EPHA8 expression is first detected around embryonic day (E) 10.5, with highest levels near the midline region of the tectum

• Reporter gene approaches:

  • Utilize EPHA8-lacZ fusion proteins that preserve the native EPHA8 expression pattern

  • Perform X-gal histochemistry to visualize expression domains

  • Expression appears in discrete regions of the hindbrain, dorsal horn of the spinal cord, and naso-lacrimal groove, decreasing after E12.5

• Axon tracing combined with immunostaining:

  • Apply anterograde tracers (e.g., DiI) to axonal origins

  • Co-stain with EPHA8 antibodies to correlate expression with specific projections

  • Compare patterns between wild-type and EPHA8-mutant mice

• Explant culture systems:

  • Culture tissue explants from regions with EPHA8 expression

  • Treat with EPHA8-blocking antibodies or ephrin-A ligands

  • Measure effects on axonal outgrowth direction and extent

• Functional perturbation approaches:

  • Generate mice homozygous for EPHA8 mutations

  • Examine aberrant axonal projections using appropriate tracers

  • Correlate phenotypes with EPHA8 expression domains and timing

• Time-lapse imaging with combined labeling:

  • Culture neurons expressing fluorescent proteins

  • Apply labeled EPHA8 antibodies to track receptor dynamics

  • Monitor growth cone behavior in response to ephrin gradients

What technical considerations are important when using biotin-conjugated EPHA8 antibodies in tumor microenvironment studies?

When investigating EPHA8 in tumor contexts:

• Multiplex immunofluorescence protocol optimization:

  • Use biotin-conjugated EPHA8 antibodies in combination with markers for:

    • Tumor-infiltrating T cells (CD3)

    • Tumor-associated macrophages (F4/80, CD163)

    • Myeloid-derived suppressor cells (CD11b, Gr-1)

  • Carefully design antibody panels to avoid spectral overlap

• Signal amplification strategies:

  • Utilize tyramide signal amplification for low-abundance targets

  • Compare direct detection versus amplified detection methods

  • Optimize streptavidin-fluorophore concentration and incubation time

• Tissue sample preparation considerations:

  • Fresh frozen versus formalin-fixed paraffin-embedded tissues require different protocols

  • For FFPE samples, optimize antigen retrieval methods (citrate buffer pH 6.0 versus EDTA pH 9.0)

  • Include proper controls for tissue autofluorescence

• Quantitative image analysis approaches:

  • Develop algorithms to quantify co-localization between EPHA8 and immune cell markers

  • Measure spatial relationships using nearest neighbor analysis

  • EPHA8 co-localizes with TAMs and MDSCs but not with T cells in certain tumor microenvironments

• Flow cytometry panel design:

  • Create multicolor panels including EPHA8 and relevant immune markers

  • Use proper compensation controls for each fluorochrome

  • The gating strategy should begin with viable cells, then identify populations using markers like CD45, CD3, CD4, CD8, and GrB

• Experimental validation in immunodeficient models:

  • Compare EPHA8 antibody effects between immunocompetent and immunodeficient (e.g., BALB/c-SCID) mice

  • Anti-EPHA8 antibody therapy shows antitumor effects in immunocompetent mice but not in immunodeficient models, suggesting immune-mediated mechanisms

How can researchers troubleshoot inconsistent results when using biotin-conjugated EPHA8 antibodies?

When facing reproducibility challenges:

• Antibody validation protocol implementation:

  • Verify epitope specificity using peptide competition assays

  • Compare results across multiple antibody lots and clones

  • Test on positive and negative control samples with known EPHA8 expression status

• Biotin conjugation quality assessment:

  • Determine biotin:antibody ratio using HABA assay

  • Measure batch-to-batch variation in conjugation levels

  • Overly high biotin loads (>20 biotin molecules per antibody) may negatively impact binding

• Storage condition optimization:

  • Evaluate antibody activity after storage at different temperatures

  • Avoid repeated freeze-thaw cycles which significantly reduce activity

  • Consider aliquoting antibodies upon receipt

• Buffer compatibility testing:

  • Some buffers may interfere with biotin-streptavidin interaction

  • Test antibody performance in different buffer systems

  • Optimize buffer components (salt concentration, pH, detergents)

• Detection system troubleshooting:

  • Compare different streptavidin conjugates (HRP, fluorophores)

  • Check for streptavidin binding to endogenous biotinylated proteins

  • Use avidin/biotin blocking kits to reduce background

• Systematic protocol optimization:

  • Titrate antibody concentration to determine optimal working dilution

  • Try different incubation conditions (time, temperature)

  • For western blotting, test various blocking agents (BSA vs. milk proteins)

  • For ELISA, recommended dilutions are typically 1:10,000, while for western blotting, 1:500 may be more appropriate