PLXNB2 Antibody， Biotin conjugated

What is PLXNB2 and why is it important in biological research?

PLXNB2 (Plexin B2) is a transmembrane protein encoded by the PLXNB2 gene that functions as a cell surface receptor primarily localized to the plasma membrane . In humans, the canonical PLXNB2 protein consists of 1838 amino acid residues with a molecular weight of approximately 205.1 kDa and undergoes post-translational modifications including glycosylation . PLXNB2 serves as a receptor for multiple semaphorin ligands, including SEMA4C, SEMA4D, and SEMA4G, mediating critical cell-cell signaling pathways involved in development, immune responses, and disease processes . Recent research has also identified PLXNB2 as the functional receptor for angiogenin (ANG), a secreted ribonuclease with context-specific roles in cell growth, survival, and regeneration across multiple cell types . The protein's involvement in diverse physiological and pathological processes, including cancer progression, neurogenesis, and hematopoietic stem cell function, makes it an important target for academic research across multiple disciplines .

What are the advantages of using biotin-conjugated PLXNB2 antibodies compared to unconjugated variants?

Biotin-conjugated PLXNB2 antibodies offer several methodological advantages over unconjugated counterparts, particularly in multi-step detection protocols and signal amplification systems. The biotin-streptavidin system provides one of the strongest non-covalent biological interactions known (Kd ≈ 10^-15 M), enabling highly sensitive detection while maintaining excellent signal-to-noise ratios in techniques like ELISA, immunohistochemistry, and flow cytometry . Biotin conjugation allows researchers to implement versatile detection strategies through secondary detection with streptavidin coupled to various reporter molecules (fluorophores, enzymes, quantum dots), providing flexibility in experimental design without requiring multiple directly-labeled primary antibodies . Additionally, biotin-conjugated antibodies facilitate signal amplification through layered streptavidin-biotin complexes, enabling detection of low-abundance PLXNB2 protein in samples where expression levels may be limited. The small size of the biotin molecule (244 Da) ensures minimal interference with antibody-antigen binding, helping preserve the specificity and affinity of the PLXNB2 antibody while adding detection capabilities .

How should biotin-conjugated PLXNB2 antibodies be stored and handled to maintain optimal activity?

Biotin-conjugated PLXNB2 antibodies require specific storage and handling protocols to preserve both antibody integrity and biotin-conjugate stability over time. For long-term storage, aliquot the antibody upon receipt and maintain at -20°C or -80°C to minimize freeze-thaw cycles, as repeated freezing and thawing can lead to protein denaturation and biotin degradation . When preparing working solutions, dilute using appropriate buffers (typically PBS containing 0.1% BSA as a carrier protein) and avoid buffers containing sodium azide when using enzymatic detection methods as azide can inhibit peroxidase activity . During experimental procedures, maintain antibody solutions at 2-8°C and use within 8 hours of preparation to prevent degradation, while also minimizing exposure to strong light which can affect both the antibody protein and the biotin conjugate . Importantly, avoid using solutions containing free biotin or biotinylated proteins that might compete for streptavidin binding sites and reduce detection efficiency in your experimental system. Always centrifuge antibody solutions briefly before opening vials to collect any solution that might be trapped in the cap or along the sides of the container .

What are the recommended protocols for using biotin-conjugated PLXNB2 antibodies in immunohistochemistry (IHC)?

For paraffin-embedded tissue sections, begin with appropriate antigen retrieval - typically heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 20 minutes, followed by cooling to room temperature . Block endogenous biotin using a commercial biotin blocking kit to prevent non-specific binding of streptavidin to endogenous biotin, especially in biotin-rich tissues like liver, kidney, and brain. Apply the biotin-conjugated PLXNB2 antibody at optimized dilutions (typically 1:100 to 1:500) in antibody diluent and incubate overnight at 4°C in a humidified chamber to ensure complete antigen access while maintaining antibody stability . Detect using streptavidin-HRP or streptavidin-AP complexes followed by appropriate chromogenic substrates (DAB for HRP, Fast Red for AP), with incubation times of 30-60 minutes at room temperature. For multiplexing with other markers, use streptavidin conjugated to spectrally distinct fluorophores and carefully plan your staining sequence to avoid cross-reactivity between detection systems . Always include both positive controls (tissues known to express PLXNB2, such as certain neural tissues or tumor samples) and negative controls (primary antibody omission and isotype controls) to validate staining specificity .

What are the optimal conditions for using biotin-conjugated PLXNB2 antibodies in flow cytometry?

When preparing cells for flow cytometry with biotin-conjugated PLXNB2 antibodies, begin with proper fixation using 2-4% paraformaldehyde for 10-15 minutes at room temperature, followed by permeabilization with 0.1% saponin or 0.1% Triton X-100 if intracellular epitopes need to be accessed . Block Fc receptors using species-appropriate blocking reagents (10% normal serum or commercial Fc receptor blocking solutions) for 15-30 minutes at room temperature to reduce non-specific binding, particularly important when analyzing immune or hematopoietic cells . For staining, use the biotin-conjugated PLXNB2 antibody at predetermined optimal concentrations (typically 1-10 μg/ml) and incubate for 30-60 minutes at 4°C protected from light, followed by washing and secondary detection with fluorophore-conjugated streptavidin (common fluorophores include PE, APC, or Alexa Fluor dyes) . When designing multi-parameter panels, carefully consider fluorophore selection to minimize spectral overlap, and always include appropriate compensation controls, fluorescence-minus-one (FMO) controls, and isotype controls to establish accurate gating strategies . For quantitative assessments, consider using calibration beads with known antibody binding capacity to convert fluorescence intensity into molecules of equivalent soluble fluorochrome (MESF) or antibody binding capacity (ABC) units for standardized reporting across experiments .

How can biotin-conjugated PLXNB2 antibodies be effectively used in ELISA applications?

For developing a sensitive ELISA using biotin-conjugated PLXNB2 antibodies, first optimize plate coating conditions with capture antibody (typically 1-10 μg/ml in carbonate/bicarbonate buffer pH 9.6) and incubate overnight at 4°C to ensure maximum binding to the plate surface . Block remaining binding sites with 1-5% BSA or commercial blocking buffer for 1-2 hours at room temperature to minimize background signal before adding samples containing PLXNB2 protein . For detection, apply the biotin-conjugated PLXNB2 antibody (for sandwich ELISA) at optimized concentration (typically 0.1-1.0 μg/ml) in appropriate diluent and incubate for 1-2 hours at room temperature with gentle agitation to ensure even distribution and binding . Follow with streptavidin-HRP (typical dilution 1:1000 to 1:5000) for 30-60 minutes at room temperature, then develop with TMB substrate and stop with 2N H₂SO₄ or 1N HCl after appropriate color development (typically 5-30 minutes) . For quantitative analysis, always include a standard curve using recombinant PLXNB2 protein at known concentrations (typically a 7-point curve with 2-fold dilutions) and calculate unknown concentrations using four-parameter logistic (4-PL) curve fitting for most accurate results across a wide dynamic range .

What protocol should be followed for Western blotting using biotin-conjugated PLXNB2 antibodies?

Begin sample preparation by lysing cells or tissues in RIPA buffer supplemented with protease inhibitors, followed by protein quantification using Bradford or BCA assay to ensure equal loading across lanes (typically 20-50 μg total protein per lane) . For PLXNB2 detection, use 6-8% polyacrylamide gels to properly resolve this high molecular weight protein (205.1 kDa), and include standard molecular weight markers to confirm target band identification . After transfer to PVDF or nitrocellulose membrane (wet transfer at 30V overnight at 4°C is recommended for large proteins), block the membrane with 5% non-fat dry milk or 5% BSA in TBST for 1 hour at room temperature before applying the biotin-conjugated PLXNB2 antibody at optimized dilution (typically 1:500 to 1:2000) in blocking buffer overnight at 4°C with gentle rocking . Following thorough washing with TBST (3-5 washes of 5-10 minutes each), incubate with streptavidin-HRP (1:2000 to 1:10,000 dilution) for 1 hour at room temperature, wash again, and detect using enhanced chemiluminescence (ECL) substrate with exposure times optimized based on signal strength . When analyzing results, be aware that PLXNB2 may show multiple bands due to glycosylation, proteolytic processing, or alternative splicing, and always validate findings with appropriate positive and negative controls to ensure specificity .

How can biotin-conjugated PLXNB2 antibodies be utilized to study the interaction between PLXNB2 and angiogenin (ANG)?

To investigate PLXNB2-ANG interactions, implement co-immunoprecipitation assays using biotin-conjugated PLXNB2 antibodies bound to streptavidin-coated magnetic beads, allowing efficient pull-down of PLXNB2-ANG complexes from cell lysates under native conditions . Proximity ligation assays (PLA) can visualize protein-protein interactions in situ by employing biotin-conjugated PLXNB2 antibodies together with ANG-specific antibodies, followed by oligonucleotide-linked streptavidin and secondary antibodies that generate fluorescent signals only when the two proteins are in close proximity (<40 nm) . For interaction dynamics, FRET (Fluorescence Resonance Energy Transfer) analysis using biotin-conjugated PLXNB2 antibodies detected with streptavidin-conjugated donor fluorophores, combined with acceptor-labeled ANG antibodies, allows measurement of real-time interaction events and conformational changes . Surface plasmon resonance (SPR) or bio-layer interferometry (BLI) assays utilizing immobilized biotin-conjugated PLXNB2 antibodies can determine binding kinetics and affinity constants between PLXNB2 and ANG with high precision . When designing these interaction studies, consider using site-directed antibodies targeting specific domains of PLXNB2 to distinguish between different binding interfaces and determine structure-function relationships in the PLXNB2-ANG signaling axis .

What approaches can be used with biotin-conjugated PLXNB2 antibodies to investigate PLXNB2's role in cancer pathogenesis?

For analyzing PLXNB2 expression patterns across tumor types and stages, employ tissue microarrays (TMAs) probed with biotin-conjugated PLXNB2 antibodies and streptavidin-HRP detection systems, allowing high-throughput screening across multiple patient samples while maintaining standardized staining conditions . Implement RNA-protein correlation studies by combining biotin-conjugated PLXNB2 antibody immunohistochemistry with RNA-scope in situ hybridization on sequential tissue sections to correlate protein expression with mRNA levels, providing insights into transcriptional versus post-transcriptional regulation mechanisms . For functional studies in cancer models, use biotin-conjugated neutralizing PLXNB2 antibodies to block interactions with ligands like ANG, followed by analysis of downstream effects on xenograft tumor growth, cancer stem cell maintenance, and metastatic potential . Investigate PLXNB2's role in cancer cell signaling by employing biotin-conjugated PLXNB2 antibodies in phospho-flow cytometry to simultaneously measure PLXNB2 expression and activation of downstream pathways including AKT and ERK phosphorylation, Rac/Cdc42 activation, and other cancer-associated signaling nodes . For translational relevance, correlate PLXNB2 expression patterns (using biotin-conjugated antibodies in pathology specimens) with clinical outcomes, treatment responses, and patient survival data to establish prognostic and predictive value .

How can biotin-conjugated PLXNB2 antibodies be applied to study neuronal functions and neurodegenerative processes?

For neuronal localization studies, implement high-resolution confocal microscopy using biotin-conjugated PLXNB2 antibodies with streptavidin-conjugated fluorophores alongside markers for specific neuronal compartments (MAP2 for dendrites, tau for axons, synaptic markers) to determine precise subcellular distribution patterns . In neuronal culture systems, utilize biotin-conjugated blocking PLXNB2 antibodies to investigate the functional consequences of disrupting PLXNB2-mediated signaling on neurite outgrowth, axon guidance, dendritic spine formation, and synaptogenesis through time-lapse imaging and morphometric analysis . For neurodegenerative disease models, apply biotin-conjugated PLXNB2 antibodies in combination with markers of neurodegeneration (amyloid-β, tau, α-synuclein) using multiplex immunofluorescence to assess potential roles in pathological processes and disease progression . Implement in vivo studies using intracerebroventricular injection of biotin-conjugated PLXNB2 antibodies (neutralizing or non-neutralizing) followed by behavioral testing and histological analysis to evaluate effects on neurogenesis, neuroprotection, and cognitive function in animal models of neurological disorders . For translational studies, examine post-mortem human brain tissues from neurodegenerative disease patients using biotin-conjugated PLXNB2 antibodies to establish disease-specific expression patterns and correlations with pathological features, providing insights into potential therapeutic interventions targeting the PLXNB2-ANG axis .

What methodologies can be employed with biotin-conjugated PLXNB2 antibodies to study hematopoietic stem cell biology and leukemia?

For hematopoietic stem and progenitor cell (HSPC) isolation, implement fluorescence-activated cell sorting (FACS) protocols using biotin-conjugated PLXNB2 antibodies with streptavidin-fluorophore detection alongside established HSPC markers (CD34, CD38, CD90, CD45RA) to identify and purify PLXNB2-expressing subpopulations for functional analyses . In colony-forming assays, pre-treat isolated HSPCs with biotin-conjugated neutralizing PLXNB2 antibodies to block interactions with ANG and other ligands, then assess effects on colony formation capacity, differentiation potential, and self-renewal properties using serial replating experiments . For leukemic stem cell (LSC) studies, compare PLXNB2 expression patterns between normal HSPCs and patient-derived LSCs using biotin-conjugated PLXNB2 antibodies in flow cytometry and immunofluorescence microscopy to identify disease-specific alterations in expression or localization . Employ xenotransplantation models by injecting AML cells pre-treated with biotin-conjugated blocking PLXNB2 antibodies into immunodeficient mice, followed by tracking of leukemia initiation, progression, and response to conventional therapies to establish the role of PLXNB2 signaling in disease maintenance . For mechanistic studies, combine biotin-conjugated PLXNB2 antibody-based cell sorting with RNA-seq and ATAC-seq to identify PLXNB2-dependent transcriptional networks and chromatin accessibility changes that drive self-renewal programs in normal and leukemic stem cells .

What are common issues encountered when using biotin-conjugated PLXNB2 antibodies and how can they be resolved?

High background signal in IHC/ICC may result from insufficient blocking of endogenous biotin, which can be addressed by implementing a specific biotin/avidin blocking step before antibody incubation or by using alternative detection systems in biotin-rich tissues . Non-specific binding can occur due to Fc receptor interactions or charge-based binding, which should be mitigated by incorporating appropriate blocking agents (normal serum, commercial Fc blockers) and optimizing salt concentration in wash and diluent buffers . Loss of signal intensity over time might indicate biotin-conjugate degradation, requiring proper storage conditions (aliquoting, -20°C storage, minimal freeze-thaw cycles) and potentially the addition of stabilizing proteins like BSA (0.1-1%) to antibody solutions . Cross-reactivity with non-target proteins can compromise specificity, necessitating validation through multiple methods (Western blot, IHC with knockout/knockdown controls) and potentially pre-absorption with recombinant PLXNB2 protein to confirm binding specificity . Signal variability between experiments may stem from inconsistent streptavidin reagent quality or degradation, requiring preparation of fresh working solutions for each experiment and potentially standardizing with calibration beads or internal reference samples across experimental runs .

How can researchers validate the specificity and sensitivity of biotin-conjugated PLXNB2 antibodies?

Conduct comprehensive specificity testing using cell lines with confirmed PLXNB2 expression versus PLXNB2 knockout or knockdown models, comparing signal patterns across multiple techniques (Western blot, IHC, flow cytometry) to ensure consistent target recognition . Perform peptide competition assays by pre-incubating the biotin-conjugated PLXNB2 antibody with excess recombinant PLXNB2 protein or immunizing peptide before application to samples, with elimination of specific signal confirming target specificity . Evaluate cross-reactivity with other plexin family members (especially closely related PLXNB1 and PLXNB3) using recombinant proteins or cell lines expressing single plexin variants to determine the degree of selectivity within this protein family . Implement orthogonal validation by comparing detection patterns using multiple antibodies targeting different epitopes of PLXNB2, with consistent localization and expression patterns supporting specific recognition . For quantitative applications, establish detection limits and dynamic range by creating standard curves with recombinant PLXNB2 protein titrations, determining the lowest detectable concentration and the linear range of measurement to guide experimental design and data interpretation .

What is the experimental data on PLXNB2 binding partners and signaling pathways?

PLXNB2 functions as a high-affinity receptor for multiple ligands, with binding affinity measurements showing Kd values in the nanomolar range for semaphorins SEMA4C (Kd ≈ 5-15 nM), SEMA4D (Kd ≈ 1-10 nM), and SEMA4G (Kd ≈ 10-30 nM) . Recent proteomic studies have identified angiogenin (ANG) as a novel binding partner, with surface plasmon resonance measurements indicating strong binding (Kd ≈ 20-50 nM) to the extracellular domain of PLXNB2, representing a previously uncharacterized signaling axis with implications across multiple biological systems . Activation of PLXNB2 triggers multiple downstream signaling cascades, including RhoGTPase signaling (primarily through Rac and Cdc42 activation), PI3K/AKT pathway stimulation (with phosphorylation at Ser473/Thr308), and ERK1/2 MAPK pathway activation (phosphorylation at Thr202/Tyr204), which collectively regulate cytoskeletal dynamics, cell motility, survival, and proliferation . In neuronal contexts, PLXNB2 signaling has been shown to modulate growth cone dynamics through localized RhoGTPase activity, with quantitative analysis demonstrating 2.5-3.5-fold increases in active Rac1 within 5 minutes of ligand stimulation, followed by cytoskeletal reorganization and directional growth responses . Interactome studies using proximity labeling approaches have identified over 50 potential PLXNB2-interacting proteins, including transmembrane receptors (MET, ErbB2), cytoskeletal regulators (filamin A, α-actinin), and signaling adaptors (Grb2, SHC), suggesting PLXNB2 functions within larger signaling complexes to integrate multiple cellular inputs .

What protocols exist for studying PLXNB2 in cancer research models?

PLXNB2 expression analyses across neural tissues have demonstrated dynamic regulation during development, with quantitative PCR and protein quantification showing highest expression in neurogenic regions (ventricular/subventricular zones, dentate gyrus) during periods of active neurogenesis, followed by gradual reduction but maintained expression in mature neurons . Knockout studies in animal models have revealed that complete PLXNB2 deficiency results in severe neural tube defects and embryonic lethality, while conditional and inducible knockouts show more subtle phenotypes including aberrant neuronal migration, reduced dendritic complexity (20-30% reduction in Sholl analysis measurements), and altered synaptic density (15-25% decrease in excitatory synapse markers) . In neurodegenerative disease contexts, PLXNB2 has emerged as a critical mediator of ANG-induced neuroprotection, with in vitro studies demonstrating that biotin-conjugated blocking PLXNB2 antibodies significantly reduce neuronal survival under stress conditions (oxidative stress, excitotoxicity) by 40-60% compared to control antibodies, highlighting the importance of this signaling axis in neuronal resilience . Time-course experiments following neuronal injury have shown rapid upregulation of both PLXNB2 and ANG in peri-injury zones (2-4 fold increase within 24-48 hours), with co-localization patterns suggesting coordinated action during regenerative responses and functional recovery . Translational studies using post-mortem samples from patients with neurodegenerative diseases have identified altered PLXNB2 expression patterns, including significant reductions in affected brain regions of Alzheimer's disease patients (30-50% decrease in hippocampus) and abnormal subcellular distribution in motor neurons of ALS patients, suggesting potential involvement in disease pathophysiology and highlighting this pathway as a therapeutic target .