efnb2a Antibody

What is Ephrin-B2 and why is it significant in research?

Ephrin-B2 (EFNB2) is a transmembrane protein encoded by the EFNB2 gene with a reported amino acid length of 333 and an expected molecular mass of 36.9 kDa. It functions as a ligand for Eph receptors, a family of receptor tyrosine kinases crucial for migration, repulsion, and adhesion during neuronal, vascular, and epithelial development . Ephrin-B2 is involved in bidirectional signaling—the pathway downstream of the receptor is called forward signaling, while the pathway downstream of the ephrin ligand is referred to as reverse signaling .

Ephrin-B2 is significant in research due to its essential roles in:

• Cardiovascular development and angiogenesis

• Neural crest cell migration and development

• Somite patterning during embryogenesis

• Synaptic plasticity in the nervous system

• Viral entry mechanisms (acts as a receptor for henipaviruses like Nipah virus)

The protein may also be known by alternative names including EPLG5, HTKL, Htk-L, LERK5, HTK ligand, and LERK-5 .

What applications are commonly supported by commercial Ephrin-B2 antibodies?

Commercial Ephrin-B2 antibodies support multiple experimental applications, as summarized in the following table:

When selecting antibodies for these applications, researchers should consider the validation data provided by manufacturers and published literature demonstrating successful application in similar experimental contexts .

How should researchers optimize Western blot protocols for Ephrin-B2 detection?

Optimizing Western blot protocols for Ephrin-B2 detection requires attention to several critical parameters:

Sample preparation:

• Use RIPA or NP-40 buffer with protease and phosphatase inhibitors for efficient membrane protein extraction

• For phosphorylation studies, include sodium orthovanadate to inhibit phosphatases

Electrophoresis and transfer:

• Use 10-12% SDS-PAGE gels for optimal separation

• PVDF membranes are recommended for transmembrane proteins like Ephrin-B2

• Consider semi-dry transfer or overnight wet transfer at low voltage for efficient membrane protein transfer

Detection conditions:

• Primary antibody concentration: Typically 1 μg/mL (range: 0.1-2 μg/mL) depending on the specific antibody

• Incubation: Overnight at 4°C for optimal binding

• Expected band size: Approximately 40-45 kDa, though glycosylation may result in higher apparent molecular weights (47-50 kDa)

Controls and troubleshooting:

• Include positive control lysates (mouse embryo tissue or neuronal cell lines like SH-SY5Y)

• For phosphorylation studies, compare phosphorylated vs. non-phosphorylated samples (e.g., before/after stimulation)

• Pre-adsorption with immunizing peptide can confirm antibody specificity

What strategies improve reproducibility when working with Ephrin-B2 antibodies?

Ensuring reproducible results with Ephrin-B2 antibodies requires systematic approaches to antibody validation and experimental design:

Antibody validation strategies:

• Cross-validation with multiple antibodies targeting different epitopes

• Testing in knockout/knockdown models as negative controls

• Peptide competition assays to confirm specificity

• Batch testing and documentation to monitor lot-to-lot variations

Experimental considerations:

• Standardize sample collection and processing protocols

• Establish consistent fixation methods for immunostaining (fixation can affect epitope availability)

• Document detailed protocols including blocking conditions, antibody concentrations, and incubation times

• Include appropriate positive and negative controls in every experiment

Data analysis approaches:

• Implement quantitative methods for signal analysis

• Use consistent normalization strategies

• Maintain detailed records of antibody sources, catalog numbers, and lot information

• Consider blind analysis to reduce bias in subjective assessments

How can researchers distinguish between forward and reverse Ephrin-B2 signaling?

Differentiating between forward and reverse Ephrin-B2 signaling requires specialized experimental approaches:

For forward signaling analysis (Eph receptor-expressing cells):

• Monitor Eph receptor phosphorylation using phospho-specific antibodies

• Track downstream effectors such as Rho GTPases, focal adhesion kinase, or MAPK pathways

• Use EphB receptor mutants that can bind Ephrin-B2 but lack kinase activity

• Employ specific inhibitors (e.g., PI 3-kinase inhibitor LY29004 or MEK-1/2 inhibitors) to block specific pathways

For reverse signaling analysis (Ephrin-B2-expressing cells):

• Track Ephrin-B2 tyrosine phosphorylation using phospho-specific antibodies (e.g., anti-phospho Y316)

• Examine SH2/SH3 adapter protein recruitment to the Ephrin-B2 cytoplasmic domain

• Utilize Ephrin-B2 mutants lacking cytoplasmic tyrosine phosphorylation sites

• Assess PDZ-dependent signaling pathways through co-immunoprecipitation studies

Experimental systems:

• Co-culture systems with cells expressing either Eph receptors or Ephrin-B2

• In vivo models with cell-type specific knockout of either the receptor or ligand

• Stimulation with clustered soluble Eph-Fc or Ephrin-Fc fusion proteins

What approaches are effective for studying Ephrin-B2's role in neural crest cell development?

Investigating Ephrin-B2's function in neural crest cells requires multi-dimensional approaches:

Genetic manipulation strategies:

• Conditional knockout using neural crest-specific Cre lines (e.g., Nav1.8-Cre)

• Temporal control using inducible systems (CreERT2)

• Generation of chimeric embryos to assess cell-autonomous effects

Expression analysis methods:

• Immunofluorescence co-labeling with neural crest markers (Sox10, FoxD3)

• In situ hybridization for efnb2 mRNA expression

• RT-PCR analysis to detect Eph receptor expression in neural crest populations

• Single-cell RNA-seq to identify subpopulations expressing Ephrin-B2

Functional assessment:

• Migration assays to evaluate neural crest movement

• Lineage tracing combined with Ephrin-B2 manipulation

• Assessment of derivative tissues (craniofacial structures, peripheral nervous system)

• Live imaging of neural crest migration in Ephrin-B2 mutants

Research has demonstrated that loss of ephrin-B2 leads to defects in populations of cranial and trunk neural crest cells, highlighting its importance in early embryonic development .

What methodological approaches can evaluate Ephrin-B2 phosphorylation status?

Assessment of Ephrin-B2 phosphorylation requires specialized techniques:

Direct detection methods:

• Phospho-specific antibodies targeting known phosphorylation sites (e.g., Y316)

• Immunoprecipitation followed by Western blot with anti-phosphotyrosine antibodies

• Phos-tag SDS-PAGE to enhance separation of phosphorylated from non-phosphorylated forms

• Mass spectrometry for comprehensive identification of phosphorylation sites

Functional studies:

• Mutation of key phosphorylation sites (e.g., tyrosine residues) to assess their functional importance

• Use of kinase inhibitors to block phosphorylation events and confirm pathway specificity

• Comparison of wild-type versus phosphorylation-deficient Ephrin-B2 in rescue experiments

In vivo approaches:

• Generation of knock-in mice expressing phosphorylation site mutants

• Phosphorylation analysis following physiological stimulation (e.g., after formalin injection in pain models)

• Correlation of phosphorylation status with functional outcomes

Research has shown that NR2B phosphorylation in spinal cord is regulated by Ephrin-B2 signaling in certain inflammatory pain models, demonstrating the functional relevance of these phosphorylation events .

How can researchers engineer specific Ephrin-B2 variants for studying receptor selectivity?

Engineering Ephrin-B2 variants with modified receptor selectivity involves strategic approaches:

Mutagenesis strategies:

• Deep mutational scanning to identify residues critical for specific receptor interactions

• Structure-guided mutagenesis targeting the G-H binding loop of EFNB2

• Creation of chimeric proteins with domains from different ephrins

Key residues for manipulation:

• D62Q mutation enhances specificity for viral glycoproteins while reducing Eph receptor binding

• Modifications at the base of the G-H binding loop affect receptor selectivity

• Mutations in the phenylalanine hinge (F113) can alter conformational states

Validation approaches:

• Surface plasmon resonance to measure binding kinetics with different receptors

• Cell-based binding assays with flow cytometry readout

• Functional assays to assess signaling outcomes with modified ligands

Combinatorial mutations (e.g., D62Q-Q130L-V167L) can create Ephrin-B2 variants with minimal binding to Eph receptors while maintaining interactions with viral glycoproteins, potentially useful for therapeutic applications .

What viral entry studies can be performed using Ephrin-B2 antibodies?

Ephrin-B2 antibodies enable diverse approaches to studying viral entry mechanisms:

Binding and competition assays:

• Antibody blocking experiments to prevent viral attachment

• Flow cytometry to quantify virus binding to Ephrin-B2-expressing cells

• ELISA-based competition assays with recombinant viral proteins

Microscopy approaches:

• Immunofluorescence co-localization of Ephrin-B2 with viral particles

• Live-cell imaging of viral entry in the presence of blocking antibodies

• Super-resolution microscopy to visualize virus-receptor interactions

Functional studies:

• Viral infection assays in cells with antibody pre-treatment

• Assessment of viral entry kinetics with receptor-specific antibodies

• Combination of antibodies targeting different epitopes to map the viral binding interface

Ephrin-B2 serves as a cell entry receptor for several henipaviruses including Nipah virus, and understanding these interactions has implications for developing antiviral strategies .

How should researchers approach Ephrin-B2 antibody validation across different species?

Cross-species validation of Ephrin-B2 antibodies requires systematic assessment:

Sequence analysis:

• Alignment of Ephrin-B2 sequences across species to identify conserved epitopes

• Mapping of antibody epitopes to assess theoretical cross-reactivity

• Analysis of post-translational modification sites that might affect antibody recognition

Experimental validation:

• Western blot testing using recombinant proteins or tissue lysates from multiple species

• Immunohistochemistry on fixed tissues from different species with known expression patterns

• Negative controls using tissues from knockout models

Optimization strategies:

• Titration of antibody concentrations for each species

• Modified fixation protocols that preserve epitopes across species

• Adjusted blocking conditions to reduce background in different tissue types

Many commercial antibodies are validated for human, mouse, and rat Ephrin-B2 , but validation for other species requires careful testing by individual researchers.

How can Ephrin-B2 antibodies be utilized in angiogenesis research?

Ephrin-B2 antibodies offer valuable tools for investigating angiogenic processes:

Expression analysis in vascular tissues:

• Immunostaining of developing vascular beds to map arterial-venous boundaries

• Co-localization with endothelial markers in tumor vasculature

• Quantification of Ephrin-B2 levels during vascular remodeling

Functional manipulation:

• Blocking antibodies to inhibit Ephrin-B2/EphB4 interactions during sprouting angiogenesis

• Tracking phosphorylation events in tip vs. stalk cells during vessel formation

• Analysis of cellular responses (migration, adhesion) following antibody treatment

In vivo applications:

• Injection of fluorescently-labeled antibodies for vascular imaging

• Therapeutic targeting of pathological angiogenesis with blocking antibodies

• Assessment of vascular normalization following treatment

Ephrin-B2 plays a central role in heart morphogenesis and angiogenesis through regulation of cell adhesion and migration, making it a valuable target for cardiovascular research .

What approaches can identify novel Ephrin-B2 binding partners beyond Eph receptors?

Discovering non-canonical Ephrin-B2 interactions requires specialized techniques:

Protein-protein interaction methods:

• Immunoprecipitation coupled with mass spectrometry

• Proximity labeling approaches (BioID, APEX)

• Protein microarray screening with recombinant Ephrin-B2

• Yeast two-hybrid screening using the Ephrin-B2 cytoplasmic domain

Validation strategies:

• Co-immunoprecipitation with candidate interactors

• Surface plasmon resonance to measure binding kinetics

• Proximity ligation assay for in situ interaction detection

• FRET or BRET assays in living cells

Functional assessment:

• siRNA knockdown of candidate interactors followed by Ephrin-B2 pathway analysis

• Co-expression studies in heterologous systems

• Structure-function analysis with domain deletion mutants

Beyond Eph receptors, Ephrin-B2 has been identified as a receptor for henipaviruses and may have additional uncharacterized binding partners involved in its diverse biological functions .