PLXNB2 Antibody，FITC conjugated

Basic Research Questions

• What is PLXNB2 and what cellular processes does it participate in?

  PLXNB2 (Plexin B2) belongs to the plexin family of proteins that serve as primary receptors for semaphorins. These proteins were originally identified as axon guidance molecules but have subsequently been implicated in angiogenesis, immunoregulation, and cancer development . PLXNB2 specifically participates in axon guidance and cell migration in response to semaphorin signaling, with notable expression in the developing central nervous system .

  The protein exists as a precursor of approximately 240 kDa that can undergo proteolytic processing to yield an alpha subunit (~170 kDa) and a beta subunit (80 kDa) . This processing is critical for its functional activity in signaling pathways. In recent research, PLXNB2 mutations have been identified in cancer of unknown primary (CUP), suggesting its potential role in tumor development and metastasis .

• What are the recommended applications for PLXNB2 antibodies in research?

  PLXNB2 antibodies have been validated for multiple research applications depending on the specific antibody formulation:

  Application	Validated Antibodies	Recommended Dilution
  Western Blot (WB)	Mouse Monoclonal (67265-1-Ig), Rabbit Polyclonal (10602-1-AP), Sheep Polyclonal (AF6836)	1:500-1:50000
  Immunohistochemistry (IHC)	Mouse Monoclonal (67265-1-Ig), Rabbit Polyclonal (10602-1-AP)	1:400-1:2000
  Immunofluorescence (IF)	Rabbit Polyclonal (10602-1-AP), FITC-conjugated	1:10-1:100
  Immunoprecipitation (IP)	Rabbit Polyclonal (10602-1-AP)	0.5-4.0 μg for 1.0-3.0 mg protein lysate
  ELISA	Mouse Monoclonal (67265-1-Ig), Rabbit Polyclonal (10602-1-AP), FITC-conjugated	Varies by assay design
  Flow Cytometry	Sheep Polyclonal (AF6836), FITC-conjugated	Assay-dependent

  The FITC-conjugated PLXNB2 antibody is particularly valuable for direct fluorescence detection in flow cytometry and immunofluorescence applications, eliminating the need for secondary antibody incubation steps .

• What are the optimal storage and handling conditions for maintaining antibody activity?

  To maintain optimal activity of PLXNB2 antibodies including FITC-conjugated variants, follow these storage and handling guidelines:

  • Store at -20°C or -80°C upon receipt

  • Avoid repeated freeze-thaw cycles that can degrade antibody quality and FITC fluorescence

  • For working solutions, store at 4°C for short-term use (1-2 weeks)

  • Most PLXNB2 antibodies are supplied in PBS buffer with 0.02% sodium azide and 50% glycerol at pH 7.3

  • FITC-conjugated antibodies should be protected from light during storage and handling to prevent photobleaching

  • For long-term storage of aliquots, -20°C is generally sufficient without the need for further aliquoting

  Always refer to the manufacturer's specific recommendations as slight variations may exist between products.

• What sample types have been validated for PLXNB2 antibody reactivity?

  PLXNB2 antibodies show reactivity with samples from various species and tissue/cell types:

  Antibody Type	Validated Species	Validated Sample Types
  Mouse Monoclonal (67265-1-Ig)	Human, Mouse, Rat	MDA-MB-231 cells, rat brain tissue, ATDC-5 cells, U2OS cells, Mouse brain tissue, MCF-7 cells, A549 cells
  Rabbit Polyclonal (10602-1-AP)	Human, Mouse, Rat	A549 cells, HEK-293 cells, HeLa cells, HepG2 cells, human brain tissue, MCF-7 cells, mouse liver tissue, mouse ovary tissue
  Sheep Polyclonal (AF6836)	Mouse	M1 mouse myeloid leukemia cell line, mouse lung tissue, mouse ovary tissue, RAW 264.7 monocyte/macrophage cell line, mouse embryo (developing CNS)
  FITC-conjugated	Human	Recombinant human samples

  When working with new sample types, preliminary validation through titration experiments is recommended to establish optimal conditions .

• What molecular weight bands should I expect when using PLXNB2 antibodies in Western blot?

  When using PLXNB2 antibodies in Western blot applications, you should expect to observe the following molecular weight bands:

  • Calculated molecular weight: 205 kDa

  • Observed molecular weights:

    • Full-length precursor: ~240 kDa

    • Alpha subunit: ~170 kDa (may appear as ~150 kDa in some systems)

    • Beta subunit: ~80 kDa

  The variation between calculated and observed molecular weights is likely due to post-translational modifications such as glycosylation. The detection of multiple bands is expected and represents different processing forms of PLXNB2 rather than non-specific binding .

Advanced Research Questions

• How can I optimize PLXNB2 immunodetection in challenging tissue samples?

  Optimizing PLXNB2 detection in challenging samples requires careful consideration of several methodological parameters:

  For immunohistochemistry:

  • Antigen retrieval is critical: TE buffer at pH 9.0 is recommended as the primary method, with citrate buffer at pH 6.0 as an alternative

  • For fixed tissues, particularly from the central nervous system, extend the antigen retrieval time to 15-20 minutes

  • For formalin-fixed samples, reduce background by using a blocking solution containing 5-10% normal serum from the same species as the secondary antibody

  • When using FITC-conjugated antibodies directly, increase the blocking step to minimize autofluorescence

  For Western blot of challenging samples:

  • Use PVDF membranes which have demonstrated superior results for PLXNB2 detection compared to nitrocellulose

  • Employ reducing conditions with immunoblot Buffer Group 1 for optimal band resolution

  • For clearer detection of the 240 kDa band, use gradient gels (4-15%) with extended separation times

  • Consider using enhanced chemiluminescence substrates with longer exposure times for weak signals

  In each case, include appropriate positive controls from validated samples (e.g., mouse brain tissue, A549 cells) to confirm assay functionality .

• What controls should I include to validate PLXNB2 antibody specificity in my experiments?

  Comprehensive validation of PLXNB2 antibody specificity requires multiple control approaches:

  Primary controls:

  • Positive tissue/cell controls: Use samples with known PLXNB2 expression (mouse brain tissue, A549 cells, HepG2 cells)

  • Negative controls: Omit primary antibody while maintaining all other steps

  • Isotype controls: For flow cytometry applications, include appropriate isotype control antibodies (e.g., Catalog # 5-001-A for sheep antibodies)

  • Absorption controls: Pre-incubate antibody with blocking peptide to verify signal reduction

  Advanced validation controls:

  • Knockdown/knockout validation: Use PLXNB2 siRNA/CRISPR to generate negative control samples

  • Multiple antibody verification: Compare staining patterns using antibodies targeting different PLXNB2 epitopes

  • Phosphatase treatment: For phosphorylation-specific detection, include controls with and without phosphatase treatment

  • Subcellular fractionation: Verify localization corresponds with known PLXNB2 distribution

  The literature indicates that PLXNB2 knockdown/knockout controls have been used in at least 3 published studies, confirming the specificity of commercially available antibodies .

• How can I design effective multiplexed experiments involving PLXNB2?

  Designing effective multiplexed experiments with PLXNB2 antibodies requires strategic planning:

  For multicolor immunofluorescence:

  • When using FITC-conjugated PLXNB2 antibody, pair with far-red fluorophores (e.g., Cy5, Alexa Fluor 647) to minimize spectral overlap

  • For co-localization studies with semaphorins or downstream effectors, use sequential staining protocols to prevent steric hindrance

  • Consider tyramide signal amplification for weak PLXNB2 signals when multiplexing

  For flow cytometry:

  • The FITC-conjugated PLXNB2 antibody works well with PE-conjugated secondary antibodies for detection of other markers

  • For multiparameter analysis, titrate the FITC-PLXNB2 antibody carefully to prevent compensation issues

  • When analyzing RAW 264.7 cells or similar macrophage populations, include CD markers to differentiate subpopulations

  For protein complex studies:

  • Co-immunoprecipitation experiments require 0.5-4.0 μg antibody per 1.0-3.0 mg protein lysate

  • Cross-linking before lysis may help preserve transient PLXNB2 interactions

  • For interaction studies with EGFR kinase (implicated in PLXNB2-associated invasiveness), use gentle detergent conditions

  Always include single-stained controls for proper compensation and antibody titration experiments to determine optimal concentrations for multiplexed detection.

• What methodologies are recommended for investigating PLXNB2 mutations in cancer research?

  Research into PLXNB2 mutations in cancer requires sophisticated methodological approaches:

  For mutation detection and characterization:

  • Next-generation sequencing (NGS) has successfully identified novel PLXNB2 mutations like G842C in cancer of unknown primary (CUP)

  • When examining PLXNB2 mutations, sequence the entire coding region as functional mutations have been identified throughout the gene (e.g., G842C, R531P, L1058S)

  • For structural analysis of mutation effects, employ in silico prediction tools that have proven valuable in predicting functional consequences

  For functional validation:

  • Patient-derived spheroid models (e.g., "agnospheres") provide faithful recapitulation of PLXNB2 mutation effects on cancer phenotype

  • When studying invasiveness, consider EGFR kinase inhibition experiments, as PLXNB2 mutations have shown EGFR kinase-dependent effects on invasion

  • For migration studies, wound healing and transwell assays with antibody detection provide quantifiable metrics

  • To assess PLXNB2 mutation effects on proliferation, BrdU incorporation followed by FITC-conjugated antibody detection allows simultaneous assessment of proliferation and protein expression

  Western blot analysis using PLXNB2 antibodies should be employed to verify protein expression levels in mutant vs. wild-type samples, with particular attention to the processing forms (240 kDa precursor vs. cleaved forms) .

• How do different fixation methods affect PLXNB2 epitope preservation and detection sensitivity?

  Fixation methodology significantly impacts PLXNB2 detection and requires careful consideration:

  Fixation Method	Impact on PLXNB2 Detection	Recommended Antibody Approach
  Formalin/PFA (4%)	Masks some epitopes; requires heat-mediated antigen retrieval	Use TE buffer pH 9.0 for retrieval; dilution 1:400-1:1600
  Methanol	Preserves cytoplasmic domain epitopes; poor for transmembrane regions	Good for detecting cleaved forms; dilution 1:200-1:800
  Acetone	Suitable for frozen sections; maintains most epitopes	Preferred for immunofluorescence; dilution 1:10-1:100
  Immersion fixation	Effective for embryonic tissues	Used successfully with sheep anti-mouse PLXNB2 at 0.6 μg/mL

  For developmental studies examining PLXNB2 in embryonic tissue, immersion fixation followed by frozen sectioning has proven effective for detecting PLXNB2 in the developing central nervous system using DAB staining methods . When using FITC-conjugated antibodies, shorter fixation times (10-15 minutes) help preserve fluorescence intensity.

  For double immunofluorescence, consider sequential fixation protocols when co-staining with markers requiring different fixation methods. The choice of fixative should be determined by the specific application and tissue type.

• What approaches should be used to investigate PLXNB2 involvement in axon guidance mechanisms?

  Investigating PLXNB2's role in axon guidance requires specialized methodological approaches:

  For developmental studies:

  • Timed embryonic tissue collection is critical, with embryonic day 13 (E13) showing strong PLXNB2 expression in the developing central nervous system

  • Immunohistochemical analysis with sheep anti-mouse PLXNB2 antibody at 0.6 μg/mL has successfully visualized PLXNB2 distribution in embryonic tissues

  • For co-localization with semaphorins, dual immunofluorescence with FITC-conjugated PLXNB2 antibodies provides direct visualization

  For functional studies:

  • Primary neuronal cultures from embryonic brain tissues allow assessment of PLXNB2 function in neurite outgrowth

  • Time-lapse microscopy with FITC-labeled antibodies enables real-time monitoring of PLXNB2 dynamics during growth cone guidance

  • Co-culture systems with semaphorin-expressing cells and neurons with labeled PLXNB2 help elucidate attraction/repulsion mechanisms

  For molecular interaction studies:

  • Co-immunoprecipitation using PLXNB2 antibodies (0.5-4.0 μg for 1.0-3.0 mg lysate) can identify binding partners

  • Proximity ligation assays with PLXNB2 antibodies provide spatial resolution of protein interactions in intact cells

  • FRET analysis using FITC-conjugated PLXNB2 antibodies paired with acceptor fluorophore-tagged semaphorins can reveal direct interactions

  When designing these experiments, consider that PLXNB2 processing (240 kDa precursor to cleaved forms) may affect functional outcomes in axon guidance contexts .

• What are the troubleshooting strategies for inconsistent PLXNB2 Western blot results?

  Addressing inconsistent Western blot results with PLXNB2 antibodies requires systematic troubleshooting:

  For weak or absent signals:

  • Verify protein extraction efficiency: PLXNB2 is a large transmembrane protein requiring efficient extraction methods such as RIPA buffer with protease inhibitors

  • Adjust protein loading: Higher protein amounts (50-80 μg/lane) may be necessary for detecting the 240 kDa band

  • Optimize transfer conditions: Extended transfer times (overnight at 30V) improve transfer of high molecular weight PLXNB2

  • Consider antibody concentration: The recommended dilution range varies widely (1:500-1:50000), requiring optimization for each sample type

  For multiple unexpected bands:

  • Verify sample degradation: Fresh preparation with protease inhibitors prevents degradation products

  • Check for isoforms: The expected bands include 240 kDa (precursor), 170 kDa (alpha subunit), and 80 kDa (beta subunit)

  • Reduce non-specific binding: Extend blocking time and use 5% BSA instead of milk for blocking

  • Assess antibody specificity: Multiple PLXNB2 antibodies target different epitopes; comparing results can identify the most specific for your application

  For inconsistent results between replicates:

  • Standardize lysate preparation: Consistent cell confluency and lysis conditions improve reproducibility

  • Control for post-translational modifications: Phosphatase inhibitors should be included if phosphorylation affects epitope recognition

  • Use validated positive controls: Mouse brain tissue, A549 cells, and HepG2 cells consistently express PLXNB2

  • Consider membrane type: PVDF membranes have demonstrated superior results for PLXNB2 detection compared to nitrocellulose

  Western blots for PLXNB2 in mutant samples should be interpreted carefully, as mutations may affect protein processing and antibody recognition .

• How can PLXNB2 antibodies be integrated into cancer stem cell research protocols?

  Incorporating PLXNB2 antibodies into cancer stem cell research requires specialized methodological considerations:

  For identification and isolation:

  • Flow cytometry with FITC-conjugated PLXNB2 antibodies can be used to identify and sort PLXNB2-expressing cancer stem cell populations

  • When analyzing cancer stem cell-enriched spheroids, dissociate completely to single cells before antibody staining to ensure complete epitope access

  • For triple marker analysis, combine FITC-PLXNB2 with far-red fluorophore-conjugated stem cell markers (CD133, CD44) to minimize spectral overlap

  For functional characterization:

  • Patient-derived xenograft (PDX) models with subsequent immunostaining for PLXNB2 can assess in vivo roles

  • "Agnosphere" models derived from cancer of unknown primary (CUP) biopsies have been successfully used to study PLXNB2 mutations

  • Invasion assays comparing wild-type versus mutant PLXNB2-expressing cancer stem cells reveal EGFR kinase-dependent invasiveness

  For mechanistic studies:

  • Co-immunoprecipitation using PLXNB2 antibodies can identify cancer stem cell-specific interaction partners

  • Chromatin immunoprecipitation (ChIP) assays following PLXNB2 activation can reveal transcriptional responses

  • Single-cell RNA sequencing paired with PLXNB2 immunophenotyping links protein expression to transcriptional programs

  Recent research has demonstrated that G842C-PLXNB2 mutation enhances cancer stem cell proliferation, tumorigenic capacity, and invasiveness in CUP models, highlighting the importance of PLXNB2 in cancer stem cell biology .