PVRL3 Antibody

What is PVRL3 and what is its biological significance?

PVRL3 (Nectin-3) is a calcium-independent immunoglobulin-like cell adhesion molecule that localizes at adherens junctions between neighboring cells. It plays a crucial role in mediating asymmetric homotypic and heterotypic cell-cell adhesions . The protein is primarily expressed in testis and placental tissues, as well as in many cell lines, including epithelial cell lines .

Biologically, PVRL3 is significant because:

• It forms part of the nectin family (containing four members, nectin-1-4)

• It participates in forming various cell-cell junctions, including cadherin-based adherens junctions in epithelial cells and fibroblasts

• It contributes to synaptic junctions in neurons and Sertoli cell-spermatid junctions in testis

• It interacts with afadin, an actin-binding protein, at cell-cell adherence junctions

• It's localized predominantly in dendrites and has been implicated in mammalian lens and ciliary body development

What types of PVRL3 antibodies are currently available for research?

Several types of PVRL3 antibodies are available for research purposes:

Note: Some antibodies target specific regions, such as the extracellular domain, which may be important depending on your research aims.

How do I select the appropriate PVRL3 antibody for my specific experiment?

When selecting a PVRL3 antibody, consider:

• Species reactivity: Ensure the antibody recognizes PVRL3 in your model organism. For example, antibody 103-A1 specifically reacts with mouse Nectin-3 but not human or rat .

• Application compatibility: Different antibodies perform optimally in different applications:

  • For Western blot: Anti-Nectin-3/PVRL3 (ANR-053) works at 1:200 dilution

  • For IHC-Paraffin: Anti-PVRL3 clone 1D1 is suitable

  • For flow cytometry: CD113 Monoclonal Antibody from Bioassay Technology Laboratory

• Epitope specificity: Some antibodies target specific domains:

  • ANR-053 targets the extracellular N-terminus (amino acids 84-97)

  • Other antibodies may target different epitopes, affecting their utility

• Validation data: Review available validation data for your specific application. For example, ANR-053 has been validated in Western blots of mouse/rat testis membranes, brain lysates, and human U-87 MG glioblastoma cell lines .

• Isotype and format: Consider the isotype (e.g., IgG2a for clone 1D1 ) and whether conjugated antibodies would benefit your application.

What are the optimal protocols for using PVRL3 antibodies in Western blot applications?

For Western blot applications using PVRL3 antibodies, follow these methodological guidelines:

• Sample preparation:

  • For tissue samples: Mouse/rat testis membranes and brain lysates have shown good results

  • For cell lines: Human U-87 MG glioblastoma and Jurkat T-cell leukemia cells express detectable levels of PVRL3

• Dilution ratios:

  • For Anti-Nectin-3/PVRL3 (extracellular) Antibody (ANR-053): Use 1:200 dilution

  • For Proteintech PVRL3 antibody (11213-1-AP): Use 1:1000-1:4000 dilution

• Blocking conditions:

  • Use standard 5% non-fat milk or BSA in TBST

• Detection considerations:

  • Expected molecular weight: While calculated at 61 kDa, PVRL3 is typically observed at 70-80 kDa due to glycosylation

  • Include appropriate positive controls (testis tissue shows reliable expression)

  • For specificity validation, use blocking peptides (such as Nectin-3/PVRL3 extracellular Blocking Peptide #BLP-NR053)

• Troubleshooting:

  • Note that some antibodies, like clone 103-A1, cannot be used for Western blot applications

  • If detecting multiple bands, this could reflect different isoforms (nectin-3 alpha, beta, and gamma)

How can PVRL3 antibodies be effectively used in immunohistochemistry studies?

For effective IHC applications with PVRL3 antibodies:

• Tissue preparation options:

  • Paraffin-embedded sections: Human prostate cancer tissue has shown good results with Proteintech antibody (11213-1-AP)

  • Frozen sections: Recommended for rat brain sections with ANR-053 antibody (1:100 dilution)

• Antigen retrieval methods:

  • For paraffin sections: Use TE buffer pH 9.0 as primary approach

  • Alternative method: Citrate buffer pH 6.0

• Optimal antibody dilutions:

  • ANR-053: Use 1:100 dilution for frozen sections

  • Proteintech 11213-1-AP: Use 1:20-1:200 dilution

• Detection systems:

  • Fluorescent detection: ANR-053 shows good results with goat anti-rabbit-AlexaFluor-488

  • Chromogenic detection: Anti-Human Nectin-3 works well with Anti-Goat HRP-DAB staining

• Tissue-specific considerations:

  • Brain tissue: PVRL3 immunoreactivity appears in cells of the pyramidal layer

  • Kidney tissue: Shows specific staining patterns with Anti-Human Nectin-3 antibody

What considerations are important when designing flow cytometry experiments with PVRL3 antibodies?

When designing flow cytometry experiments with PVRL3 antibodies:

• Cell preparation:

  • Live intact human Jurkat T-cell leukemia cells have been successfully used for cell surface detection of PVRL3

  • Ensure gentle dissociation methods to preserve membrane integrity and surface antigens

• Antibody selection:

  • Choose antibodies specifically validated for flow cytometry, such as:

    • CD113 Monoclonal Antibody from Bioassay Technology Laboratory

    • Anti-Nectin-3/PVRL3 (extracellular) Antibody for live cell surface staining

• Staining protocol optimization:

  • For live cell staining with ANR-053: Use 2.5 μg of antibody, followed by goat-anti-rabbit-FITC

  • Include appropriate controls:

    • Unstained cells

    • Secondary antibody only (e.g., cells + goat-anti-rabbit-FITC without primary antibody)

    • Isotype control antibody

• Gating strategy:

  • First gate on viable cells (using appropriate viability dye)

  • Consider co-staining with cell type-specific markers when analyzing heterogeneous populations

• Data analysis considerations:

  • Report results as percentage of positive cells and/or mean fluorescence intensity

  • For quantitative comparisons, use calibration beads to standardize fluorescence measurements

How can PVRL3 antibodies be used to study its role in Clostridium difficile toxin binding?

PVRL3 has been identified as a cellular receptor for Clostridium difficile toxin B (TcdB). To study this interaction:

• Experimental approaches:

  • Cell viability assays: Compare TcdB cytotoxicity in wild-type cells versus those with PVRL3 knockdown (via shRNA) or knockout (via CRISPR/Cas9)

  • Binding inhibition studies: Pretreat cells with anti-PVRL3 antibodies before TcdB exposure (effective at 13-27 nM concentrations)

  • Protein-protein interaction assays: Use purified PVRL3 ectodomain to pull down TcdB

  • Competitive inhibition: Preincubate TcdB with purified PVRL3 ectodomain before cell treatment (15 nM TcdB is effectively neutralized)

• Key controls and validations:

  • Include PVRL1 and PVRL2 as specificity controls (they don't inhibit TcdB cytotoxicity)

  • Use complementation with PVRL3 expression vectors to confirm specificity of knockout/knockdown effects

  • Compare antibody effects with isotype control antibodies at equivalent concentrations

• Readout methods:

  • ATP-based viability assays for long-term toxicity (18-48h)

  • LDH release assays for shorter-term cytotoxicity measurements (4.5h)

What are the common challenges in immunoprecipitation experiments with PVRL3 antibodies and how can they be addressed?

Common challenges in immunoprecipitation (IP) experiments with PVRL3 antibodies include:

• Low efficiency of precipitation:

  • Solution: Optimize antibody amounts (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate is recommended for Proteintech antibody)

  • Alternative approach: Consider using mouse testis tissue, which shows reliable PVRL3 expression for IP

• Non-specific binding:

  • Solution: Implement more stringent washing conditions or use pre-clearing steps

  • Validation method: Include appropriate negative controls (isotype control antibody or IgG from same species)

• Protein complex disruption:

  • Solution: Use gentler lysis buffers to preserve interactions with binding partners like afadin

  • Consideration: For studying PVRL3-afadin complexes, co-IP may require optimization

• Cross-reactivity concerns:

  • Solution: Validate specificity using PVRL3 knockout/knockdown samples

  • Alternative: Use blocking peptides to confirm antibody specificity

• Detection challenges post-IP:

  • Solution: Use different antibody clone for detection than was used for precipitation

  • Consideration: Be aware of the 70-80 kDa observed molecular weight (versus 61 kDa calculated)

How can PVRL3 antibodies be utilized to investigate its role in congenital ocular development disorders?

PVRL3 plays an important role in mammalian lens and ciliary body development, and has been associated with congenital ocular disorders. Research approaches include:

• Immunohistochemical analysis:

  • Target tissues: Mouse embryonic ciliary body and lens

  • Antibody selection: Use antibodies validated for developmental tissue studies

  • Co-staining approach: Combine with markers for cell junctions (E-Cadherin, ZO-1) and Nectin partners (PVRL1, Afadin)

• Expression analysis in disease models:

  • Approach: Compare PVRL3 expression in wild-type versus mutant mice (e.g., ari - anterior retinal inversion mutant)

  • Method: In situ hybridization using antisense riboprobe from PVRL3 retina EST clone (with sense riboprobe as control)

  • Visualization: Dark field microscopy with hematoxylin counterstaining

• Genetic analysis approaches:

  • Position effect studies: Investigate how chromosomal rearrangements affect PVRL3 expression

  • Regulatory element identification: Analyze regions with histone modifications (H3K4Me1, H3K27Ac) that may contain long-range control elements

• Functional studies:

  • Knockout models: Analyze ocular phenotypes in PVRL3 knockout mice

  • Cell adhesion assays: Evaluate altered cell-cell junctions in lens and ciliary body development

What methods are recommended to address inconsistent results when using different PVRL3 antibodies?

When facing inconsistent results across different PVRL3 antibodies:

• Epitope mapping and comparison:

  • Different antibodies target different epitopes (e.g., ANR-053 targets amino acids 84-97 in the extracellular N-terminus)

  • Determine which epitope regions are recognized by each antibody

  • Assess whether post-translational modifications might affect epitope accessibility

• Isoform-specific detection:

  • PVRL3 has three isoforms: nectin-3 alpha, beta, and gamma

  • Determine which isoforms each antibody detects based on epitope locations

  • Consider using RT-PCR to confirm which isoforms are expressed in your experimental system

• Cross-validation approaches:

  • Use at least two different antibodies recognizing distinct epitopes

  • Compare with genetic approaches (siRNA knockdown, CRISPR knockout)

  • Validate with recombinant expression systems

• Application-specific optimization:

  • Some antibodies are application-restricted (e.g., clone 103-A1 cannot be used for Western blot)

  • Test different fixation/permeabilization methods for IHC/IF

  • Optimize protein extraction methods for Western blot

• Species-specific considerations:

  • Some antibodies have narrow species reactivity (e.g., 103-A1 works only for mouse, not human or rat)

  • Ensure antibody validation data exists for your specific species

  • Consider sequence homology at the epitope region when switching between species

How are PVRL3 antibodies being used to study its role in cell-cell adhesion and junction formation?

PVRL3 antibodies are being utilized in several key approaches to study cell-cell adhesion and junction formation:

• Localization studies:

  • Immunofluorescence microscopy to visualize PVRL3 at adherens junctions

  • Co-localization with junction proteins (cadherins, afadin, other nectins)

  • Super-resolution microscopy to examine nanoscale organization at junctions

• Functional blocking experiments:

  • Using antibodies against the extracellular domain to disrupt trans-interactions

  • Time-lapse imaging to observe junction formation/dissolution following antibody treatment

  • Measuring barrier function or adhesion strength changes after antibody application

• Protein complex analysis:

  • Immunoprecipitation to identify PVRL3-containing protein complexes

  • Proximity ligation assays to confirm in situ interactions with binding partners

  • Mass spectrometry analysis of PVRL3-associated proteins in different cell types

• Specialized junction studies:

  • Analysis of Sertoli cell-spermatid junctions in testis, where PVRL3 is highly expressed

  • Examination of synaptic junctions in neurons

  • Investigation of heterotypic junctions formed through PVRL3-PVRL2 interactions

• Developmental context:

  • Tracing PVRL3 expression during embryonic development of tissues requiring complex junctions

  • Correlating junction formation with tissue morphogenesis using antibody staining

What are the critical controls necessary when using PVRL3 antibodies in genetic knockout or knockdown validation studies?

When using PVRL3 antibodies to validate genetic manipulations:

• Essential negative controls:

  • PVRL3 knockout tissues/cells: Complete gene knockout via CRISPR/Cas9 should eliminate specific staining

  • PVRL3 knockdown samples: shRNA or siRNA knockdown should show reduced signal proportional to knockdown efficiency

  • Blocking peptide controls: Pre-incubation of antibody with immunizing peptide should abolish specific signals

• Important positive controls:

  • Wild-type matched samples: Process simultaneously with knockout/knockdown samples

  • Rescue experiments: Re-expression of PVRL3 in knockout cells should restore antibody staining

  • Tissues with known expression: Include testis or brain samples as positive references

• Technical validation approaches:

  • Multiple antibodies: Use antibodies recognizing different epitopes to confirm results

  • Multiple detection methods: Combine protein (Western blot, IHC) with mRNA detection (RT-PCR, in situ hybridization)

  • Loading controls: Include housekeeping genes/proteins to normalize expression levels

• Specificity controls:

  • Related protein controls: Test for cross-reactivity with other nectin family members (PVRL1, PVRL2, PVRL4)

  • Isotype controls: Use matched isotype antibodies to identify non-specific binding

  • Secondary antibody-only controls: Ensure secondary antibodies don't contribute to signal

How can PVRL3 antibodies be used to investigate its potential role in motion sickness susceptibility?

Recent genetic studies have identified PVRL3 variants associated with motion sickness susceptibility . To investigate this connection:

• Genetic-molecular correlations:

  • Use PVRL3 antibodies to compare protein expression levels between individuals with different genotypes at the rs66800491 locus (3q13.13)

  • Analyze tissue-specific expression patterns in relevant tissues (inner ear, brain regions involved in balance)

• Functional studies in model systems:

  • Generate cell or animal models with the specific SNP variants

  • Use PVRL3 antibodies to assess:

    • Changes in protein localization or expression level

    • Alterations in protein-protein interactions

    • Modifications to cell adhesion properties in relevant cell types

• Structural biology approaches:

  • Combine antibody epitope mapping with structural analysis to understand how genetic variants might affect protein function

  • Use conformation-specific antibodies to detect structural changes associated with variants

• Pathway analysis:

  • Employ PVRL3 antibodies in immunoprecipitation followed by mass spectrometry to identify differential protein interactions in variant vs. wild-type contexts

  • Investigate potential connections to other balance-related genes identified in motion sickness studies (e.g., GPD2, ACO1)

• Translational applications:

  • Develop screening assays using PVRL3 antibodies to identify compounds that might modulate its function

  • Investigate potential correlation between PVRL3 protein levels and motion sickness susceptibility

What are the emerging applications of PVRL3 antibodies in cancer research?

PVRL3 antibodies are finding increasing applications in cancer research:

• Expression profiling in tumors:

  • IHC analysis of PVRL3 in various cancer types (reports of expression in human prostate cancer and glioblastoma )

  • Correlation of expression levels with clinical outcomes (e.g., association with poor prognosis in pancreatic adenocarcinoma )

  • Tissue microarray screening across multiple cancer types

• Metastasis and invasion studies:

  • Investigation of PVRL3 role in control of tight junctions during metastasis

  • Analysis of cell-cell adhesion changes during epithelial-mesenchymal transition

  • Correlation of PVRL3 expression with invasive properties

• Therapeutic target evaluation:

  • Using antibodies to block PVRL3 function in cancer cell lines

  • Testing effects on proliferation, migration, and invasion

  • Developing potential therapeutic antibodies based on research findings

• Cancer immunology applications:

  • Investigating PVRL3's role in lymphocyte transendothelial migration

  • Studying interactions between cancer cells and immune cells mediated by PVRL3

  • Exploring PVRL3 as a potential immunomodulatory target

• Biomarker development:

  • Evaluating PVRL3 as a diagnostic or prognostic biomarker

  • Developing sensitive ELISA assays using well-characterized antibodies

  • Correlation with other established cancer biomarkers

What are the recommended storage and handling procedures to maintain PVRL3 antibody performance?

To maintain optimal PVRL3 antibody performance:

• Storage conditions:

  • Store at -20°C for long-term storage

  • For reconstituted lyophilized antibodies (e.g., ANR-053):

    • Store at 4°C for up to 1 week

    • For longer periods, prepare small aliquots and store at -20°C

    • Avoid multiple freeze-thaw cycles

• Reconstitution guidelines:

  • For lyophilized antibodies (e.g., ANR-053):

    • Use double distilled water: 25 μL, 50 μL, or 0.2 mL depending on sample size

    • Allow complete dissolution before use

  • For liquid formulations:

    • Brief centrifugation before opening is recommended

• Working solution preparation:

  • Dilute in appropriate buffer just before use

  • For ANR-053, centrifuge all antibody preparations before use (10,000 × g, 5 min)

  • Store working solutions at 4°C and use within recommended timeframes

• Stability considerations:

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

  • For small volume antibodies (20 μL), some suppliers include 0.1% BSA for stability

• Safety precautions:

  • Some antibodies contain sodium azide (0.02%) as a preservative

  • Handle according to appropriate safety guidelines, as sodium azide is hazardous

What validation methods should be employed to confirm PVRL3 antibody specificity before experimental use?

To ensure PVRL3 antibody specificity:

• Genetic validation approaches:

  • Test antibody on PVRL3 knockout or knockdown samples

  • Use CRISPR/Cas9-generated knockout cell lines as negative controls

  • Rescue experiments with PVRL3 re-expression to confirm specificity

• Peptide competition assays:

  • Pre-incubate antibody with blocking peptide (e.g., Nectin-3/PVRL3 extracellular Blocking Peptide #BLP-NR053)

  • Compare staining patterns with and without peptide competition

  • Specific signals should be abolished or significantly reduced

• Cross-reactivity testing:

  • Test against related nectin family members (PVRL1, PVRL2, PVRL4)

  • Use tissues/cells with known expression patterns of each family member

  • Verify signal corresponds to expected tissue distribution of PVRL3

• Multiple antibody validation:

  • Compare results using antibodies targeting different epitopes

  • Consistent results across multiple antibodies increase confidence in specificity

  • Be aware that some antibodies may be isoform-specific

• Recombinant protein controls:

  • Test antibody against recombinant PVRL3 protein

  • Include dose-response curve to confirm signal proportionality

  • For Western blot, verify correct molecular weight (accounting for post-translational modifications)

How can researchers troubleshoot weak or non-specific signals when using PVRL3 antibodies?

When encountering weak or non-specific signals:

• For weak signals in Western blot:

  • Increase protein loading (PVRL3 may be expressed at low levels in some tissues)

  • Reduce antibody dilution (try 1:200 instead of 1:1000)

  • Extend primary antibody incubation time or temperature

  • Use more sensitive detection systems (ECL Prime or similar)

  • Enrich for membrane fractions for this transmembrane protein

• For high background in IHC/IF:

  • Optimize blocking conditions (try different blocking agents: BSA, normal serum, commercial blockers)

  • Increase blocking time or concentration

  • Reduce primary antibody concentration

  • Include additional washing steps

  • For autofluorescence issues, consider Sudan Black B treatment or spectral unmixing

• For unexpected bands in Western blot:

  • Consider post-translational modifications (observed MW is 70-80 kDa vs. calculated 61 kDa)

  • Test multiple antibodies recognizing different epitopes

  • Include positive control tissues (testis, brain) for band comparison

  • Use gradient gels for better resolution of closely migrating bands

• For inconsistent results across experiments:

  • Standardize sample preparation methods

  • Use fresh antibody aliquots to avoid freeze-thaw degradation

  • Include positive controls in each experiment

  • Maintain consistent incubation times and temperatures

  • Verify antibody lot consistency with supplier

• For species-specific issues:

  • Confirm antibody reactivity with your species (e.g., clone 103-A1 only works with mouse)

  • Consider sequence homology at the epitope region

  • Test species-specific positive control samples