TJP2 Antibody

What are the primary research applications for TJP2 antibodies?

TJP2 antibodies are validated for multiple experimental applications, with specific performance characteristics in each context:

• Western Blot (WB): Enables detection of TJP2 protein at approximately 150 kDa (observed) versus 134 kDa (calculated) molecular weight .

• Immunohistochemistry (IHC): Allows visualization of tissue localization patterns, with particularly strong signals in epithelial and endothelial intercellular junctions .

• Immunofluorescence (IF/ICC): Reveals subcellular localization, showing TJP2 distribution between cytoplasm and nucleus depending on experimental conditions .

• Immunoprecipitation (IP): Enables isolation of TJP2 protein complexes for interaction studies .

• ELISA: Provides quantitative measurement of TJP2 protein levels .

• Flow Cytometry: Allows detection and quantification of TJP2 in cell populations .

The choice of application should be guided by your specific research question and the antibody's validated performance in that application.

How should TJP2 antibodies be stored and handled to maintain optimal performance?

Proper storage and handling of TJP2 antibodies is critical for maintaining their reactivity and specificity:

• Storage temperature: Store at -20°C for long-term preservation. Some formulations can be stored at 4°C for up to one month after reconstitution .

• Aliquoting: For antibodies provided in liquid form, aliquoting prevents repeated freeze-thaw cycles. For 20 μL sizes containing 0.1% BSA, aliquoting may not be necessary for -20°C storage .

• Buffer composition: Most TJP2 antibodies are provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Reconstitution: For lyophilized antibodies, reconstitute according to manufacturer instructions, typically in sterile water or buffer solution .

• Freeze-thaw cycles: Avoid repeated freeze-thaw cycles as they can lead to protein denaturation and reduced antibody activity .

Following these storage guidelines will help maintain antibody reactivity and experimental reproducibility.

What species reactivity can be expected from commercially available TJP2 antibodies?

TJP2 antibodies show various reactivity profiles across species, with important considerations for cross-species applications:

When selecting a TJP2 antibody, consider both the validated reactivity (confirmed experimentally) and predicted cross-reactivity (based on sequence homology). For novel applications in untested species, preliminary validation is strongly recommended.

What are the optimal antibody dilutions for different experimental applications?

Dilution optimization is crucial for achieving specific signal with minimal background. Recommended starting dilutions for TJP2 antibodies vary by application:

Always perform a dilution series during initial antibody characterization, as optimal dilutions can vary based on sample type, target abundance, and detection method. For reproducibility, maintain consistent antibody concentrations across experimental replicates.

What antigen retrieval methods are recommended for TJP2 immunohistochemistry?

Appropriate antigen retrieval is critical for optimal TJP2 detection in fixed tissues:

• Heat-induced epitope retrieval (HIER): Most commonly recommended for TJP2 detection

  • EDTA buffer (pH 8.0): Frequently cited as optimal for TJP2 antibodies

  • Citrate buffer (pH 6.0): Alternative approach that works with some antibodies

• Enzymatic antigen retrieval:

  • Used for some applications, particularly immunocytochemistry

  • Typically involves 15-minute treatment with specific enzymatic retrieval reagents

• Protocol variations:

  • Boiling tissue sections for 20 minutes followed by cooling

  • Steaming sections for 20 minutes

The optimal retrieval method should be empirically determined for each antibody and tissue type. Some antibodies (e.g., Proteintech 18900-1-AP) specifically recommend TE buffer pH 9.0 with alternative options noted .

How can I validate TJP2 antibody specificity for my experimental system?

Comprehensive validation of TJP2 antibody specificity should include multiple approaches:

• Positive controls:

  • Cell lines with known TJP2 expression (HepG2, HEK-293, HeLa, MCF-7, A431)

  • Tissue samples with established TJP2 expression patterns (liver, kidney)

• Negative controls:

  • Immunoglobulin isotype controls at equivalent concentrations

  • Samples without primary antibody incubation

  • Knockdown/knockout models where TJP2 expression is reduced/eliminated

• Molecular weight verification:

  • Confirm detection at expected molecular weight (calculated: 134 kDa; observed: 150-160 kDa)

  • Multiple bands may indicate isoforms, post-translational modifications, or degradation products

• Peptide competition:

  • Pre-incubation with immunizing peptide should abolish specific signal

  • Dot blot analysis can confirm phospho-specific antibody selectivity

• Cross-validation with alternative antibodies:

  • Use multiple antibodies targeting different epitopes of TJP2

  • Consistent results across antibodies increases confidence in specificity

Thorough validation is particularly important when studying novel experimental systems or when antibody performance is critical for data interpretation.

How should I approach TJP2 subcellular localization studies using immunofluorescence?

TJP2 exhibits complex subcellular localization patterns that can vary by cell type and physiological conditions:

• Fixation considerations:

  • Paraformaldehyde (PFA) fixation (typically 4%) preserves membrane architecture

  • Methanol fixation may enhance nuclear epitope accessibility

  • Optimization of fixation time is critical (typically 10-20 minutes)

• Permeabilization protocol:

  • Triton X-100 (0.1-0.5%) is commonly used for TJP2 detection

  • Permeabilization strength affects detection of membrane-associated versus nuclear TJP2

• Co-localization markers:

  • Membrane/junction markers: Claudin-1 (CLDN1), F-actin

  • Cytoskeletal markers: β-tubulin

  • Nuclear markers: DAPI for nuclear counterstaining

• Localization interpretation:

  • Wild-type TJP2 typically expresses in both cytoplasm and nucleus

  • Mutations can alter this distribution pattern - for example, the TJP2 c.1202A>G mutation causes enhanced nuclear localization

  • Environmental stressors can trigger TJP2 translocation between compartments

• Image acquisition parameters:

  • Use appropriate filter sets for fluorophores

  • Confocal microscopy provides superior resolution for membrane localization studies

  • Z-stack imaging may be necessary to fully capture junction architecture

The research by Zhang et al. demonstrates that TJP2 localization can shift dramatically with genetic mutations, emphasizing the importance of careful subcellular localization analysis in disease-related studies .

What controls should be included when using TJP2 antibodies in phosphorylation studies?

When studying TJP2 phosphorylation, particularly at sites like Ser978, specific controls are essential:

• Phospho-specific antibody validation:

  • Dot blot analysis comparing phospho-peptide versus non-phospho-peptide recognition

  • Validation using phosphatase treatment of samples to confirm specificity

  • Titration of antibody concentration (recommended working concentration: 0.6 μg/ml for phospho-TJP2-S978)

• Signaling pathway controls:

  • Positive controls: Treatments known to induce TJP2 phosphorylation

  • Negative controls: Inhibitors of relevant kinases

  • Time-course experiments to capture phosphorylation dynamics

• Functional validation:

  • Correlation with GSK-3β activity, which is regulated by TJP2 and affects its Ser9 phosphorylation

  • Confirmation of phosphorylation effects on TJP2 function (e.g., localization, protein interactions)

• Technical considerations:

  • Use phosphatase inhibitors during sample preparation

  • Include positive controls from cell types with known phosphorylation patterns

  • Consider parallel analysis with total TJP2 antibodies to normalize phospho-signal

Phosphorylation studies are particularly relevant given research showing that TJP2 regulates GSK-3β phosphorylation, which in turn affects cellular apoptosis susceptibility .

How can TJP2 antibodies be used to investigate cytoskeletal interactions in tight junction research?

TJP2 plays a critical role in connecting tight junctions to the actin cytoskeleton, which can be investigated using specialized approaches:

• Co-immunoprecipitation studies:

  • Use TJP2 antibodies for immunoprecipitation followed by blotting for cytoskeletal proteins

  • Recommended IP protocols: 0.5-4 μg antibody per 200-400 μg cell extract

  • Target interactions: F-actin, cytoskeletal regulatory proteins

• Co-localization analysis:

  • Dual immunofluorescence with TJP2 and cytoskeletal markers

  • F-actin staining is particularly informative as TJP2 knockdown results in dispersed F-actin and disordered microfilaments

  • β-tubulin staining reveals microtubule organization, though this appears less affected by TJP2 depletion

• Functional perturbation experiments:

  • siRNA knockdown of TJP2 followed by cytoskeletal analysis

  • Two validated siRNAs (1573 and 1673) have been shown to effectively reduce TJP2 expression

  • Document changes in:

    • F-actin organization and microfilament structure

    • Cell morphology and junction integrity

    • Expression of cytoskeleton-related genes

• Gene expression analysis:

  • Following TJP2 knockdown, monitor expression changes in cytoskeleton-related genes

  • Key cytoskeletal pathway genes affected by TJP2 depletion include PDGFRB, PIK3CB, ITGA2, TMSB4X, GSN, PIK3R3, and ITGAV

  • KEGG pathway analysis reveals significant enrichment for "regulation of actin cytoskeleton" in TJP2-depleted cells

Research by Zhang et al. demonstrated that TJP2 knockdown significantly impacts actin cytoskeleton organization but has minimal effects on microtubule structure, providing important insights into TJP2's specific role in cytoskeletal regulation .

How should I interpret unexpected molecular weight variations when detecting TJP2 by Western blot?

TJP2 often shows discrepancies between calculated and observed molecular weights in Western blot:

• Expected versus observed weights:

  • Calculated molecular weight: 131-134 kDa

  • Observed molecular weight: 150-160 kDa

• Potential explanations for variations:

  • Post-translational modifications: Phosphorylation (e.g., at S978) , glycosylation, ubiquitination

  • Isoform expression: Multiple TJP2 isoforms exist with varying molecular weights

  • Sample preparation: Differences in lysis buffers, denaturation conditions

  • Gel system variations: Different SDS-PAGE percentages (5-20% gradients are commonly used)

• Methodological considerations for accurate sizing:

  • Use appropriate molecular weight markers spanning 100-250 kDa range

  • Include positive control samples (e.g., HepG2, HeLa, MCF-7 cell lysates)

  • Consider running gradient gels (5-20%) for better resolution of high molecular weight proteins

  • Optimize transfer conditions for large proteins (longer transfer times, lower methanol percentages)

• Troubleshooting steps for molecular weight concerns:

  • Verify antibody specificity through knockdown/knockout controls

  • Test multiple validated TJP2 antibodies targeting different epitopes

  • Examine sample treatment effects (heating time/temperature, reducing conditions)

  • Consider specialized techniques (e.g., pulse-field gel electrophoresis) for very large proteins

The consistently higher observed molecular weight across multiple studies suggests that post-translational modifications significantly contribute to TJP2's apparent size in SDS-PAGE.

What factors could lead to contradictory results when studying TJP2 function using antibody-based techniques?

Several factors can contribute to contradictory results in TJP2 studies:

• Antibody-specific considerations:

  • Epitope differences: Antibodies targeting different TJP2 regions may yield varying results

    • N-terminal antibodies: May detect specific isoforms

    • C-terminal antibodies: Common target region (e.g., ABIN2775643, CAB0594)

    • Phospho-specific antibodies: Detect only phosphorylated forms (e.g., S978)

  • Cross-reactivity: Some antibodies may recognize related proteins (e.g., TJP1/ZO-1, TJP3/ZO-3)

  • Batch-to-batch variability: Especially relevant for polyclonal antibodies

• Experimental system variations:

  • Cell type differences: TJP2 function varies between epithelial, endothelial, and other cell types

  • Culture conditions: Confluence level significantly impacts tight junction formation

  • Sample preparation: Differences in fixation, lysis, or extraction protocols

• Biological complexities:

  • Nuclear vs. membrane localization: TJP2 functions in both compartments

  • Context-dependent function: TJP2 interacts with multiple signaling pathways

  • Compensatory mechanisms: Other junction proteins may compensate for TJP2 alterations

• Resolution strategies:

  • Use multiple antibodies targeting different epitopes

  • Include appropriate positive and negative controls

  • Clearly document experimental conditions

  • Validate findings with complementary methods (e.g., genetic manipulation, recombinant protein studies)

  • Consider the biological context when interpreting results

Research by Zhang et al. demonstrates how TJP2 mutations can have seemingly contradictory effects - the c.1202A>G mutation increases TJP2 expression and nuclear localization, while the c.2668-11A>G mutation results in truncated protein production .

How do I reconcile TJP2 antibody staining patterns across different tissue types?

TJP2 expression and localization patterns vary considerably across tissues, requiring careful interpretation:

• Tissue-specific expression patterns:

  • Epithelial tissues: Strong membrane localization at cell-cell junctions

  • Endothelial tissues: Junction-specific localization

  • Neuronal tissues: Both cytoplasmic and nuclear patterns observed

  • Testicular tissue: Strong cytoplasmic and membranous positivity in seminiferous ducts

  • Liver tissue: Important for tight junction integrity; mutations linked to PFIC4

• Technical factors affecting staining patterns:

  • Fixation method: Formalin vs. frozen section preparation

  • Antigen retrieval: EDTA (pH 8.0) vs. citrate buffer (pH 6.0)

  • Detection system: Chromogenic vs. fluorescent detection

  • Antibody concentration: Titration needed for each tissue type

• Biological interpretation challenges:

  • Distinguishing specific from non-specific staining

  • Correlation with known biology of each tissue type

  • Integration with functional data

• Validation approaches:

  • Compare patterns across multiple antibodies

  • Include tissue-specific positive and negative controls

  • Correlate with mRNA expression data

  • Confirm specificity using genetic models when possible

Immunohistochemistry data from multiple studies show that TJP2 exhibits distinct patterns in tissues like liver, kidney, intestine, and brain, reflecting its diverse roles in different tissue contexts .

How can TJP2 antibodies be utilized in disease mechanism studies, particularly for PFIC4?

TJP2 antibodies are valuable tools for investigating Progressive Familial Intrahepatic Cholestasis Type 4 (PFIC4), a genetic disorder caused by TJP2 mutations:

• Diagnostic applications:

  • Immunohistochemical analysis of liver biopsies to assess TJP2 expression and localization

  • Correlation of staining patterns with specific TJP2 variants

  • Comparison with other tight junction proteins (e.g., CLDN1) to evaluate junction integrity

• Mechanistic investigations:

  • Protein expression analysis: Western blotting to quantify TJP2 levels in patient-derived cells

  • Subcellular localization studies: Immunofluorescence to track normal versus mutant TJP2 localization

  • Functional assays: Combined with barrier function measurements to correlate TJP2 alterations with functional defects

  • Pathway analysis: Identification of dysregulated pathways (e.g., actin cytoskeleton regulation, TGFβ signaling)

• Model system applications:

  • Cell models: Using antibodies to validate TJP2 CRISPR/siRNA knockdown models

  • Animal models: Characterizing TJP2 expression in disease models

  • Patient-derived materials: Analyzing TJP2 in patient biopsies or iPSC-derived hepatocytes

• Therapeutic development support:

  • Screening for compounds that restore proper TJP2 localization

  • Monitoring TJP2 expression/function in response to experimental therapies

  • Identifying pathway-specific interventions based on TJP2-associated gene expression changes

Zhang et al.'s research demonstrated that TJP2 antibodies were crucial for characterizing how novel pathogenic variants (c.1202A>G and c.2668-11A>G) affect TJP2 expression, localization, and downstream pathway regulation in PFIC4 models .

What methodological considerations are important when using TJP2 antibodies to study apoptosis and cell proliferation?

TJP2 has significant roles in regulating apoptosis and cell proliferation, which can be studied using TJP2 antibodies with specific methodological considerations:

• Experimental design for proliferation studies:

  • Combine TJP2 detection with proliferation markers (Ki-67, BrdU incorporation)

  • Monitor effects of TJP2 manipulation (overexpression, knockdown) on cell growth curves

  • Consider cell type-specific effects (e.g., HepG2 vs. LO2 cells show similar responses to TJP2 knockdown)

  • Standard assays: Cell counting kit-8 (CCK-8) shows significant proliferation inhibition at 48h and 72h post-TJP2 knockdown

• Apoptosis pathway investigation:

  • TJP2-p53 axis: TJP2 knockdown increases p53 protein expression

  • GSK-3β regulation: TJP2 overexpression decreases GSK-3β Ser9 phosphorylation, increasing GSK-3β activity and apoptosis susceptibility

  • Detection methods: Flow cytometry for quantitative apoptosis measurement following TJP2 manipulation

• Technical considerations:

  • Antibody selection: Choose antibodies validated for fixed/permeabilized cells when combining with apoptosis/proliferation assays

  • Timing: TJP2 effects on proliferation are time-dependent (significant at 48h and 72h)

  • Controls: Include both positive controls (known apoptosis inducers) and negative controls

• Pathway analysis integration:

  • Combine TJP2 antibody studies with analysis of:

    • Apoptosis pathway components (BCL2 family proteins, caspases)

    • Cell cycle regulators

    • Transcriptome analysis (DEG identification)

  • KEGG pathway enrichment analysis revealed that TJP2 knockdown affects genes involved in apoptosis pathways

Research by Walsh et al. and Zhang et al. established critical links between TJP2, GSK-3β signaling, and apoptosis regulation that have implications for both hearing loss and liver diseases .

How can phospho-specific TJP2 antibodies contribute to signal transduction research?

Phospho-specific antibodies targeting TJP2 modifications (e.g., Ser978) provide unique insights into signaling pathways:

• Signal pathway mapping:

  • Kinase identification: Determining which kinases phosphorylate TJP2 at specific sites

  • Pathway integration: Understanding how TJP2 phosphorylation connects to broader signaling networks

  • Temporal dynamics: Tracking phosphorylation changes in response to stimuli

• Technical approaches:

  • Western blotting: Quantifying phosphorylation levels in response to treatments

  • Immunofluorescence: Determining if phosphorylation affects subcellular localization

  • Phospho-peptide specificity: Validation using dot blot analysis with phospho and non-phospho peptides

  • Recommended concentration: 0.6 μg/ml for phospho-TJP2-S978 detection

• Biological significance exploration:

  • Functional consequences: Does phosphorylation alter TJP2's ability to:

    • Interact with junction proteins

    • Bind to the actin cytoskeleton

    • Regulate nuclear processes

  • Disease relevance: Are phosphorylation patterns altered in pathological conditions?

• Experimental design considerations:

  • Include appropriate phosphatase inhibitors during sample preparation

  • Compare phospho-TJP2 with total TJP2 levels

  • Consider time-course experiments to capture signaling dynamics

  • Include pathway inhibitor controls to validate specificity

Phospho-specific antibodies like anti-phospho-TJP2(S978) (RayBiotech) provide tools for investigating how post-translational modifications regulate TJP2 function in normal physiology and disease states.