PLXNB2 Antibody, HRP conjugated

Basic Research Questions

• What is the optimal detection method for PLXNB2 using HRP-conjugated antibodies?

PLXNB2 (Plexin-B2) antibodies conjugated with HRP are primarily optimized for ELISA applications as indicated in multiple validation studies. When using ELISA for detection, follow these methodological approaches:

• Use freshly prepared samples whenever possible

• Employ a standard curve with recombinant PLXNB2 protein for quantification

• Follow recommended dilution ratios: typically 1:5000-1:50000 for Western blot applications

• For ELISA applications, optimize antibody concentration in preliminary experiments

• Include appropriate negative controls (isotype control antibodies) and positive controls (tissues known to express PLXNB2, such as brain tissue)

While primarily validated for ELISA, some researchers have successfully used HRP-conjugated PLXNB2 antibodies in Western blot applications with enhanced chemiluminescence detection systems.

• What are the expected molecular weights when detecting PLXNB2 in Western blots?

PLXNB2 detection requires careful interpretation of molecular weight patterns due to post-translational modifications and potential processing of the protein:

The difference between calculated (205 kDa) and observed (240 kDa) molecular weight is attributed to glycosylation and other post-translational modifications. When conducting Western blot analysis, use reducing conditions with Immunoblot Buffer Group 1 for optimal detection .

• What is the species reactivity of PLXNB2 Antibody, HRP conjugated?

The species reactivity of commercially available PLXNB2 antibodies varies by manufacturer and clone:

• The HRP-conjugated antibody described in search result shows reactivity with Human samples

• Some PLXNB2 antibodies show cross-reactivity with mouse samples (approximately 25% cross-reactivity with recombinant mouse Plexin B2)

• Human and mouse PLXNB2 share 82% amino acid identity over positions 20-1160

When studying non-human species, validation experiments are essential to confirm cross-reactivity. For experiments requiring mouse-specific detection, dedicated anti-mouse PLXNB2 antibodies are available .

• How should samples be prepared for optimal PLXNB2 detection?

For optimal detection of PLXNB2 using HRP-conjugated antibodies, follow these sample preparation guidelines:

• For cell lysates: Lyse cells in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, and protease inhibitor cocktail

• For tissue samples: Homogenize in RIPA buffer supplemented with protease inhibitors

• Include phosphatase inhibitors when studying PLXNB2 signaling

• For Western blot: Denature samples at 95°C for 5 minutes in reducing sample buffer

• For immunohistochemistry: Use paraformaldehyde fixation followed by antigen retrieval with TE buffer pH 9.0

• For flow cytometry: Use gentle cell dissociation methods to preserve membrane integrity

Avoid repeated freeze-thaw cycles of antibody as indicated in storage recommendations .

Intermediate Research Questions

• How can researchers differentiate between cleaved subunits of PLXNB2 in experimental systems?

Distinguishing between the alpha and beta subunits of PLXNB2 requires strategic experimental approaches:

• Use antibodies targeting different epitopes: Choose antibodies recognizing the N-terminal region (aa 20-466, Semaphorin domain) for alpha subunit detection and antibodies against the C-terminal region for beta subunit detection

• Perform subcellular fractionation: The beta subunit is membrane-associated while the alpha subunit may dissociate

• Use non-reducing versus reducing conditions: Under non-reducing conditions, the alpha and beta subunits may remain non-covalently associated

• Perform immunoprecipitation with subunit-specific antibodies followed by Western blot

• For confirmation, use PLXNB2 knockout cell lines as negative controls, as demonstrated in Western blot validation studies

The alpha subunit (~170 kDa) and beta subunit (~80 kDa) remain non-covalently linked after proteolytic processing, which occurs in the extracellular domain .

• What controls should be included when using PLXNB2 Antibody, HRP conjugated?

Robust experimental design requires appropriate controls:

Western blot validation using PLXNB2 knockout HeLa cell line demonstrates antibody specificity, where the expected band at approximately 170 kDa is detected in parental HeLa cells but absent in the knockout line .

• What methodologies are optimal for studying PLXNB2-ANG interactions?

To study the interaction between PLXNB2 and Angiogenin (ANG), consider these methodological approaches:

• Co-immunoprecipitation (Co-IP): Pull down PLXNB2 and probe for ANG or vice versa to detect physical interaction

• Surface Plasmon Resonance (SPR): Quantify binding kinetics (reported Kd = 0.74 nM between ANG and PLXNB2)

• ELISA: Assess binding between recombinant proteins

• Proximity Ligation Assay: Visualize protein interactions in situ

• Nuclear translocation assays: Detect ANG localization dependent on PLXNB2

• Functional assays: Measure cell proliferation, AKT/ERK phosphorylation, and rRNA transcription in the presence of PLXNB2 antibodies or PLXNB2 knockdown

Studies have shown that PLXNB2 mediates ANG-induced activities including nuclear translocation, stress granule localization, rRNA transcription, and tiRNA production, making these readouts valuable for interaction studies .

• How does PLXNB2 expression vary across cell types and how should this inform experimental design?

PLXNB2 expression varies significantly across cell lineages, requiring tailored experimental approaches:

• Highest expression in cancer tissues (10^-10 times higher than normal tissues)

• Elevated expression in stem cells compared to differentiated cells

• High expression in neuronal cells, particularly cerebellar granule cells

• Present in hematopoietic lineages, with highest levels in stem cells compared to whole bone marrow or lineage-positive cells

• Expressed in monocytic-myeloid lineage cells including dendritic cells and macrophages

For experiments with primary cells, consider cell type-specific isolation protocols and verify PLXNB2 expression levels. When studying low-expressing cells, sensitivity can be enhanced by using signal amplification techniques such as tyramide signal amplification for immunohistochemistry.

Advanced Research Questions

• How can PLXNB2 Antibody, HRP conjugated be used to investigate PLXNB2's role in regulating Rac and Cdc42 activity?

PLXNB2's role in regulating Rac and Cdc42 activity can be investigated using these methodological approaches:

• GTPase activity assays: Measure levels of GTP-bound Rac and Cdc42 in cells with and without PLXNB2 inhibition/knockdown

• Pull-down assays with GST-PAK-CRIB domain to capture active Rac/Cdc42

• Time-course experiments following cytokine stimulation (e.g., M-CSF) to track temporal changes in GTPase activity

• Combination of PLXNB2 antibody blockade with GTPase inhibitors to determine pathway interactions

• Immunofluorescence to visualize subcellular localization of active GTPases in relation to PLXNB2

• What approaches can be used to study the functional relationship between PLXNB2 and its semaphorin ligands?

Study the PLXNB2-semaphorin interactions using these methodologies:

• Domain mapping: Use truncated constructs to identify specific interaction domains (Sema domain aa 20-466 is critical)

• Cell rounding assays: Measure morphological changes as functional readouts of PLXNB2-semaphorin signaling

• Cell invasion assays: Utilize Matrigel-coated Transwell filters to assess invasion capabilities

• Co-expression studies: Express PLXNB2 with semaphorins or GTPases like Rnd3 to observe interaction effects

• Site-directed mutagenesis: Generate PLXNB2 mutants to identify key residues for semaphorin binding

• RNA interference: Use siRNA-mediated knockdown to assess functional consequences

Research has shown that PLXNB2 enhances Rnd3-induced cell rounding and inhibits invasion. Functional interactions can be visualized through co-staining of PLXNB2 and FLAG-tagged Rnd3 proteins, with actin filaments detected using fluorescently-conjugated phalloidin .

• How can PLXNB2 antibodies be used to investigate its role in cancer progression?

To investigate PLXNB2's role in cancer progression:

• Immunohistochemical profiling: Compare PLXNB2 expression across cancer stages and grades

• Survival correlation: Analyze PLXNB2 expression in relation to patient outcomes (elevated PLXNB2 correlates with reduced survival in prostate cancer, glioma, and breast cancer patients)

• Functional blockade: Use antibodies against the ANG binding site (aa 424-441) to inhibit tumor growth

• Cell proliferation assays: Measure cancer cell growth following PLXNB2 antibody treatment or PLXNB2 knockdown

• In vivo xenograft models: Assess tumor growth inhibition using anti-PLXNB2 antibodies

• RNA processing analysis: Measure 47S rRNA transcription and tiRNA production as downstream readouts

Studies have shown that antibodies generated against the ANG binding site on PLXNB2 can inhibit established xenograft tumors in vivo, suggesting therapeutic potential. The monoclonal antibody mAb17 has demonstrated complete blocking of nuclear translocation of ANG and abolishment of angiogenic activity .

• What methods are available for studying PLXNB2's role in neuronal development and function?

For investigating PLXNB2's neuronal functions:

• Neurite outgrowth assays: Measure neurite length and branching in P19 cells treated with PLXNB2 antibodies

• Apoptosis assays: Assess protection against serum starvation-induced apoptosis in neuronal cells

• ANG nuclear translocation: Visualize subcellular localization under growth conditions

• Stress granule formation: Monitor ANG localization to stress granules under stress conditions

• Genetically modified mouse models: Use conditional PLXNB2 knockout in specific neuronal populations

• Electrophysiology: Measure functional consequences of PLXNB2 manipulation on neuronal activity

PLXNB2 is expressed on the surface of P19 cells, and antibodies against PLXNB2 (e.g., mAb17) inhibit P19 cell proliferation and diminish the protective activity of ANG against serum starvation-induced apoptosis . PLXNB2 mediates ANG-induced neurogenesis and neuroprotection, suggesting potential therapeutic implications for neurodegenerative diseases like ALS and Parkinson's disease .

• How can researchers design experiments to study PLXNB2's function in hematopoietic stem cells?

For studying PLXNB2's role in hematopoietic stem cells:

• Flow cytometry: Identify and isolate PLXNB2-expressing hematopoietic stem/progenitor cells

• Colony formation assays: Assess self-renewal capacity following PLXNB2 manipulation

• Competitive transplantation: Compare reconstitution potential of wild-type versus PLXNB2-deficient stem cells

• Gene expression analysis: Measure pro-self-renewal transcripts in response to PLXNB2-ANG signaling

• Conditional gene deletion: Use Cre-loxP systems for lineage-specific PLXNB2 deletion

• Leukemia progression models: Assess the impact of PLXNB2 inhibition on leukemic stem cell function