PLXNB2 Antibody

What is PLXNB2 and what cellular functions does it regulate?

PLXNB2 (Plexin B2) is a transmembrane receptor for semaphorin family proteins with a calculated molecular weight of 205 kDa, though it is observed at 240 kDa in Western blots due to post-translational modifications . PLXNB2 functions primarily include:

• Axon guidance and neuronal migration

• Stress response regulation in the amygdala

• Modulation of glial cell function (enriched in astrocytes and microglia)

• T-cell migration to germinal centers for optimizing antibody responses

• Inflammatory pain regulation and microglial-mediated wound healing after spinal cord injury

Research has revealed that biallelic variants in PLXNB2 cause a recessive syndrome with amelogenesis imperfecta and sensorineural hearing loss as core features, with variable intellectual disability, ocular disease, ear developmental abnormalities and lymphoedema also reported .

What applications are PLXNB2 antibodies validated for?

PLXNB2 antibodies have been validated for multiple experimental applications:

These applications have been confirmed through multiple publications, with data showing reactivity in human, mouse, and rat samples .

How should PLXNB2 antibodies be stored to maintain optimal performance?

For optimal performance of PLXNB2 antibodies:

• Store at -20°C

• Most preparations are stable for one year after shipment when stored properly

• Antibodies are typically stored in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• Aliquoting is generally unnecessary for -20°C storage

• Some preparations (e.g., 20μl sizes) may contain 0.1% BSA

Avoid repeated freeze-thaw cycles by making working aliquots if frequent use is anticipated. Before each use, briefly centrifuge the vial to collect solution at the bottom.

How can I distinguish between different PLXNB2 isoforms in Western blot analysis?

When analyzing PLXNB2 via Western blot, multiple bands may be observed:

• Full-length PLXNB2 precursor: ~240 kDa

• Alpha subunit: ~170 kDa

• Beta subunit: ~80 kDa

This banding pattern occurs because "plexin B2 precursor is 240 kDa and can be cleaved into alpha subunit (~170-kDa) and beta subunit (80 kDa)" . To properly distinguish these forms:

• Use gradient gels (4-15%) to resolve high molecular weight proteins

• Include positive control lysates (e.g., mouse brain tissue, MDA-MB-231 cells, or A549 cells)

• Use reducing conditions with immunoblot buffer group 1

• Extend transfer time to ensure complete transfer of high molecular weight proteins

• Block with 5% non-fat milk or BSA in TBST

The observed molecular weight can vary depending on the cell/tissue type and physiological conditions, so comparative analysis across samples is recommended.

What are the optimal conditions for detecting PLXNB2 in brain tissue sections?

For optimal PLXNB2 detection in brain tissue sections by IHC:

Recommended Protocol:

• Antigen Retrieval:

  • Primary option: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

• Antibody Dilutions:

  • For polyclonal antibody (10602-1-AP): 1:400-1:1600

  • For monoclonal antibody (67265-1-Ig): 1:500-1:2000

• Tissue-Specific Considerations:

  • Mouse brain tissue shows strong positive signal

  • For human brain tissue, longer incubation times may be necessary

• Detection System:

  • DAB (3,3'-diaminobenzidine) provides optimal visualization

  • Counterstain with hematoxylin for contrast

PLXNB2 expression is particularly prominent in developing central nervous system tissue, as demonstrated in mouse embryo (13 d.p.c.) sections .

How can I use PLXNB2 antibodies to study stress responses in neuropsychiatric conditions?

Research has established PLXNB2's role in stress responses, particularly in schizophrenia. When designing experiments:

• Tissue Selection:

  • Focus on amygdala tissue, where PLXNB2 expression negatively correlates with stress perception

  • Examine glial cells (astrocytes and microglia) where PLXNB2 is enriched

• Functional Blocking Experiments:

  • Use functional blocking monoclonal antibodies (e.g., mAb-102) to inhibit amygdaloid PLXNB2

  • This approach has been shown to induce anxiety, amygdaloid enlargement, and microglial ramification in mouse models

• Flow Cytometry for Cell-Type Specific Analysis:

  • Use multi-color flow cytometry with the following markers:

    • Plxnb2-PE (#145903)

    • CD11b-BV421 (#101251) for microglia

    • CD45-BV650 (#103151) for immune cells

    • Glast-APC (#130-123-555) for astrocytes

• Analytical Approaches:

  • Correlate PLXNB2 expression with neuroimaging data (amygdala size)

  • Analyze stress perception scores in relation to PLXNB2 levels

  • Examine microglial morphology changes following PLXNB2 inhibition

This approach has revealed that "PLXNB2 regulates amygdala-dependent stress responses" .

How do I validate the specificity of new PLXNB2 antibodies in my experimental system?

Thorough validation of PLXNB2 antibodies requires:

• Positive Control Testing:

  • Western blot: Use tissues/cells with known PLXNB2 expression (brain tissue, MDA-MB-231, A549, HepG2 cells)

  • IHC: Mouse brain tissue or human tonsillitis tissue show reliable positivity

• Knockout/Knockdown Validation:

  • Implement PLXNB2 siRNA or CRISPR-based knockout models

  • Compare antibody signal between wildtype and KO/KD samples

  • Multiple publications have successfully used this approach

• Cross-Reactivity Testing:

  • Test in multiple species if working across species boundaries

  • For example, AF6836 shows approximately 9% cross-reactivity with recombinant human Plexin B2 in direct ELISAs

• Application-Specific Controls:

  • For flow cytometry: Include isotype control antibody (e.g., Catalog # 5-001-A)

  • For IP: Include non-immune IgG controls

• Batch Validation:

  • Each new lot should be tested against a reference lot or established positive control

  • Document band patterns and intensities across applications

What are the considerations when studying PLXNB2 mutant variants in disease models?

When investigating PLXNB2 mutations in disease contexts:

• Mutation-Specific Approaches:

  • Different PLXNB2 mutations affect distinct protein domains: extracellular (p.Ile805Phe and p.Asp750Asn) vs. intracellular portions (p.Gly1537Ser)

  • Some mutations (e.g., G842C) may enhance stem cell proliferation and invasiveness

• Structural Analysis:

  • I-TASSER-MTD modeling can help predict effects of mutations, though accuracy may be limited by lack of homologous structures

  • Variants may alter binding to semaphorins via the sema domain, affect homodimerization, or impact catalytic activity via the GAP domain

• Functional Assessments:

  • Compare wildtype vs. mutant PLXNB2 in cell rounding assays

  • Analyze invasion potential using Matrigel-coated Transwell filters

  • Examine downstream signaling pathways, particularly those involving EGFR kinase

• Antibody Selection:

  • Choose antibodies that target epitopes distant from the mutation site

  • For mutations affecting protein processing, use antibodies recognizing different regions to assess changes in PLXNB2 cleavage patterns

Genetic background significantly influences PLXNB2 mutation phenotypes, consistent with observations in knockout mouse models where homozygosity was lethal on one genetic background but viable on another .

What are the optimal protocols for extracting and preserving PLXNB2 for immunodetection?

For optimal PLXNB2 extraction and preservation:

• Tissue Preparation:

  • Fresh tissue: Gently homogenize through 70μm cell strainers on ice

  • Fixed tissue: Perform antigen retrieval with TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0

• Protein Extraction:

  • Use mild lysis buffers containing protease inhibitors

  • For membrane proteins, include 0.5-1% non-ionic detergents (e.g., NP-40 or Triton X-100)

  • Sonication may help release membrane-bound PLXNB2

• Sample Processing for Flow Cytometry:

  • Block homogenates in PBS+10% rat serum for 1 hour with gentle rotation at 4°C

  • Dilute fluorescent antibody markers in 200μl PBS+1%FBS

  • Incubate for 1 hour at 4°C with light protection

• Preservation Methods:

  • For longer-term storage, aliquot samples to avoid freeze-thaw cycles

  • Store protein lysates at -80°C

  • For tissue sections, post-fixation should be brief to prevent epitope masking

How do I troubleshoot non-specific binding when using PLXNB2 antibodies?

When encountering non-specific binding:

• Optimize Blocking Conditions:

  • Increase blocking time (1-2 hours)

  • Try different blocking agents (BSA, normal serum, commercial blockers)

  • For brain tissue, add 0.1-0.3% Triton X-100 to reduce background

• Antibody Dilution Optimization:

  • Test a range of dilutions beyond the recommended range

  • For WB: Try 1:5000-1:50000 for 67265-1-Ig

  • For IHC: Try 1:400-1:1600 for 10602-1-AP

• Washing Protocols:

  • Increase number and duration of washes

  • Use 0.1% Tween-20 in TBS/PBS for more stringent washing

  • For IHC/IF, use agitation during washing steps

• Controls to Identify Non-Specific Binding:

  • Include secondary antibody-only controls

  • Use pre-immune serum at the same concentration as primary antibody

  • If available, use PLXNB2 knockout/knockdown samples

• Tissue-Specific Considerations:

  • For brain tissue: Add extra blocking steps with avidin/biotin if using biotin-based detection

  • For highly vascularized tissues: Pre-block with mouse IgG to reduce endogenous Ig binding

What considerations should be made when designing multi-color flow cytometry panels including PLXNB2?

For multi-color flow cytometry including PLXNB2:

• Fluorophore Selection:

  • PLXNB2-PE has been successfully used in published protocols

  • Consider spectral overlap with other markers such as CD11b, CD45, and Glast

  • Use compensation controls for each fluorochrome

• Marker Combinations for Cell Identification:

  • For microglia: CD11b-BV421 (#101251), CD45-BV650 (#103151)

  • For astrocytes: Glast-APC (#130-123-555)

  • For activation status: MHCII-BV711 (#107643)

• Sample Preparation:

  • Ensure single-cell suspensions without aggregates

  • Titrate antibodies to determine optimal staining concentration

  • Include unstained, single-stained, and FMO (Fluorescence Minus One) controls

• Gating Strategy:

  • First gate on viable cells (using viability dye)

  • Identify cell populations (microglia: CD11b⁺CD45low; astrocytes: Glast⁺)

  • Then analyze PLXNB2 expression within these populations

• Analysis Approaches:

  • Compare median fluorescence intensity (MFI) of PLXNB2 between conditions

  • Analyze percentage of PLXNB2⁺ cells within each population

  • Correlate PLXNB2 expression with activation markers

This approach has been successfully used to demonstrate that "Plxnb2 was enriched in astrocytes and microglia and CUS reduced its expression in astrocytes" .

How can PLXNB2 antibodies be utilized in cancer research, particularly for invasion studies?

PLXNB2 has emerging roles in cancer biology, with antibodies being valuable tools for investigation:

• Invasion Assays:

  • Use Matrigel-coated Transwell filters to measure cellular invasion

  • Compare wild-type versus PLXNB2-overexpressing or mutant cells

  • Functional blocking antibodies can be used to inhibit PLXNB2 activity

• Molecular Mechanisms:

  • PLXNB2 mutations (e.g., G842C) can enhance cancer stem cell proliferation and EGFR kinase-dependent invasiveness

  • Study interactions with Rnd3, which when bound to PLXNB2 induces cell rounding and inhibits invasion

• Technical Approaches:

  • Combine IF with phalloidin staining to visualize actin filaments and cell morphology changes

  • Measure cell spread area following PLXNB2 manipulation

  • Use knockdown-validated antibodies to confirm specificity

• Biomarker Potential:

  • Analyze PLXNB2 expression in tumor samples using IHC

  • Validated dilution for human cancer tissues: 1:400-1:1600

  • Positive signal has been confirmed in human liver cancer, breast cancer, and gliomas tissue

Research has shown that "G842C-PLXNB2 is a novel genetic change enhancing CUP stem cell proliferation, tumorigenic capacity, and EGFR kinase-dependent invasiveness" .

What are the latest approaches for studying PLXNB2 in neuroimmune interactions?

Recent research highlights PLXNB2's role in neuroimmune interactions:

• Glial Cell Analysis:

  • PLXNB2 is enriched in astrocytes and microglia

  • CUS (chronic unpredictable stress) reduces PLXNB2 expression specifically in astrocytes

  • Use cell type-specific markers in conjunction with PLXNB2 antibodies:

    • Astrocytes: Glast

    • Microglia: CD11b, CD45

    • Activation state: MHCII

• Functional Blocking Studies:

  • Intra-amygdaloid injection of PLXNB2 blocking antibody (mAb-102) induces:

    • Anxiety behavior

    • Amygdaloid enlargement

    • Microglial ramification

• Mechanistic Analysis:

  • Analyze downstream signaling pathways following PLXNB2 activation/inhibition

  • Consider interactions with semaphorins and their effects on immune cell migration

  • Study PLXNB2's role in guiding T cell migration to germinal centers

• Clinical Correlations:

  • Examine PLXNB2 expression in psychiatric disorders (e.g., schizophrenia)

  • Correlate expression with stress perception and amygdala size

  • Investigate genetic variants that may alter PLXNB2 function in disease contexts