TJP2 Antibody, Biotin conjugated

What is TJP2 and what is its cellular function?

TJP2 (Tight Junction Protein 2), also known as Zona Occludens 2 or ZO2, is a protein encoded by the TJP2 gene in humans. It belongs to a family of membrane-associated guanylate kinase (MAGUK) homologs involved in organizing epithelial and endothelial intercellular junctions . TJP2 plays a critical role in establishing and regulating epithelial barrier function through its interaction with the actin cytoskeleton, maintaining the structure and function of tight junctions . Additionally, it functions in adherens junctions and acts as a positive regulator of RANKL-induced osteoclast differentiation, potentially via mediating downstream transcriptional activity . The protein interacts with claudins through its PDZ1 domain, which is essential for tight junction formation and stability.

What are the key applications for TJP2 antibodies in research?

TJP2 antibodies are versatile tools employed across multiple research applications:

• Western Blotting (WB): For detecting TJP2 protein expression in cell or tissue lysates, with bands typically appearing at approximately 150 kDa despite a calculated molecular weight of about 131 kDa

• Immunohistochemistry (IHC): For examining TJP2 localization in tissue sections, revealing cytoplasmic and membranous patterns in various cell types

• Immunocytochemistry (ICC)/Immunofluorescence (IF): For visualizing TJP2 distribution in cultured cells with high resolution

• ELISA: For quantitative measurement of TJP2 in experimental samples

These applications enable researchers to investigate TJP2's expression patterns, subcellular localization, and functional roles in various biological contexts.

Why choose biotin-conjugated TJP2 antibodies specifically?

Biotin-conjugated TJP2 antibodies offer several distinct advantages in research applications:

• Enhanced Signal Amplification: The biotin-streptavidin system provides significant signal amplification due to the exceptionally strong biotin-streptavidin interaction (Kd ≈ 10^-15 M), thereby increasing detection sensitivity for low-abundance targets

• Versatile Detection Options: Biotin conjugates can be paired with various streptavidin-reporter molecules (HRP, AP, fluorophores), providing flexibility in experimental design

• Multi-step Detection Compatibility: Particularly valuable in protocols requiring amplification stages or complex detection schemes

• Low Background in Optimized Systems: When properly blocked for endogenous biotin, these conjugates can provide excellent signal-to-noise ratios

For TJP2 detection specifically, biotin conjugates are particularly useful when studying tissues where TJP2 expression might be lower or when conducting multiplexed assays requiring signal amplification.

What is the recommended protocol for Western blotting with TJP2 antibodies?

Optimizing Western blotting for TJP2 requires attention to its high molecular weight (~150 kDa). The following protocol has been validated for effective TJP2 detection:

Sample Preparation and Gel Electrophoresis:

• Load 30 μg of protein per lane under reducing conditions

• Use 5-20% gradient SDS-PAGE gels for optimal resolution of high molecular weight proteins

• Run at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours

Transfer and Blocking:

• Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

• Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

Antibody Incubation:

• For primary detection, dilute TJP2 antibody to 0.5 μg/mL and incubate overnight at 4°C

• For biotin-conjugated antibodies, use appropriate streptavidin-HRP conjugate

• For standard antibodies, use anti-species HRP-conjugated secondary antibody at 1:5000 dilution

• Incubate secondary detection reagent for 1.5 hours at room temperature

Washing and Detection:

• Wash membrane with TBS-0.1%Tween 3 times, 5 minutes each, between incubations

• Develop using enhanced chemiluminescence detection systems

This protocol consistently yields specific TJP2 detection across multiple cell lines, including human RT4, A431, HepG2, MCF-7, U2OS, HeLa, PC-3, and rat cell lines .

What are the optimal conditions for immunohistochemistry using TJP2 antibodies?

Successful immunohistochemical detection of TJP2 requires specific protocol optimization:

Tissue Preparation and Antigen Retrieval:

• Process paraffin-embedded tissue sections at 4-5 μm thickness

• Perform heat-mediated antigen retrieval in EDTA buffer (pH 8.0) for optimal epitope exposure

Blocking and Antibody Application:

• Block sections with 10% goat serum (or serum matching secondary antibody species)

• For primary detection, apply TJP2 antibody at 2 μg/ml concentration

• Incubate overnight at 4°C for maximal sensitivity

Detection Systems:

• For biotin-conjugated antibodies: Apply streptavidin-HRP followed by DAB chromogen

• For standard antibodies: Use HRP-conjugated secondary antibody followed by DAB

• Counterstain with hematoxylin for structural context

Expected Results:

• In cerebral cortex: Strong cytoplasmic positivity in glial cells

• In colon tissue: Moderate cytoplasmic and membranous positivity in glandular cells

• In liver and kidney: Junction-associated and cytoplasmic localization patterns

This protocol consistently produces specific TJP2 staining across multiple tissue types while minimizing background.

How should researchers approach immunofluorescence with TJP2 antibodies?

Immunofluorescence with TJP2 antibodies requires attention to specific protocol details:

Sample Preparation:

• For cultured cells: Grow to 70-90% confluence on coverslips

• Fix with 4% paraformaldehyde (10-15 minutes) or ice-cold methanol (10 minutes)

• For tissue sections: Use fresh frozen or properly fixed paraffin sections

Antigen Retrieval and Blocking:

• For paraffin sections: Perform enzyme antigen retrieval for 15 minutes

• Block with 10% goat serum to minimize non-specific binding

• Include 0.1-0.3% Triton X-100 for membrane permeabilization

Antibody Application:

• Apply TJP2 antibody at 5 μg/mL concentration

• Incubate overnight at 4°C for optimal binding

• For biotin-conjugated antibodies: Follow with fluorophore-conjugated streptavidin

• For standard antibodies: Apply fluorophore-conjugated secondary antibody (1:500 dilution)

• Incubate secondary detection for 30-60 minutes at 37°C

Visualization:

• Counterstain nuclei with DAPI for orientation

• Mount with anti-fade medium to prevent photobleaching

• Image using appropriate fluorescence filters

This approach provides specific visualization of TJP2 with minimal background, allowing detailed analysis of its subcellular distribution.

How does biotin-conjugated TJP2 antibody perform in multiplexed staining?

Multiplexing with biotin-conjugated TJP2 antibodies requires special considerations to achieve reliable results:

Potential Challenges:

• Endogenous biotin in tissues can create background signals

• Limited fluorophore options when using streptavidin-based detection systems

• Potential cross-reactivity when using multiple biotin-conjugated primary antibodies

Optimal Multiplexing Strategies:

• Implement avidin/biotin blocking steps to minimize endogenous biotin background

• Use sequential detection protocols rather than simultaneous application when using multiple biotin-conjugated antibodies

• Combine biotin-conjugated TJP2 antibody with directly labeled antibodies for other targets

• Consider tyramide signal amplification (TSA) for multi-color experiments with biotin conjugates

Validated Co-staining Combinations:

• TJP2 with cytoskeletal markers (actin, tubulin) for junction-cytoskeleton interactions

• TJP2 with other junction proteins (claudins, occludin) for comprehensive junction analysis

• TJP2 with cell type-specific markers for analyzing junction composition in heterogeneous tissues

When properly optimized, multiplexed staining with biotin-conjugated TJP2 antibodies can reveal important contextual information about tight junction organization and function.

What are the tissue-specific considerations for TJP2 antibody applications?

TJP2 antibody performance varies across tissue types, requiring specific optimization strategies:

Epithelial Tissues (Colon, Lung, Kidney):

• Strong membrane localization at cell-cell contacts

• Moderate cytoplasmic and membranous positivity in glandular cells

• Optimization focus: Preserve junction integrity during processing

Neural Tissues (Brain, Cerebral Cortex):

• Strong cytoplasmic positivity in glial cells

• Higher background potential due to complex tissue architecture

• Optimization focus: Extended washing steps and careful antibody titration

Hepatic Tissue:

• TJP2 localizes to bile canaliculi and hepatocyte junctions

• Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) provides optimal results

• Optimization focus: Manage high endogenous biotin and peroxidase activity

Tissue-Specific Protocol Adjustments:

• Adjust antigen retrieval duration and method based on tissue density

• Modify blocking protocols to address tissue-specific background issues

• Optimize antibody concentration for each tissue type

Understanding these tissue-specific considerations ensures consistent and reliable TJP2 detection across diverse experimental systems.

How can researchers validate TJP2 antibody specificity?

Rigorous validation of TJP2 antibody specificity is essential for reliable research outcomes:

Essential Validation Approaches:

• Multiple Detection Methods Comparison:

  • Compare results across applications (WB, IHC, IF) to verify consistent patterns

  • Confirm detection of the expected 150 kDa band in Western blot

• Positive and Negative Controls:

  • Test on known TJP2-expressing samples (A431, HepG2, MCF-7, U2OS cells)

  • Include antibody omission controls while maintaining all other steps

• Genetic Validation:

  • Verify reduced signal in TJP2 knockdown/knockout systems

  • Confirm increased signal in TJP2 overexpression models

• Peptide Competition:

  • Pre-incubate antibody with immunizing peptide before application

  • A specific antibody will show significantly reduced signal after peptide blocking

• Cross-Reactivity Assessment:

  • Test against related proteins (TJP1/ZO-1, TJP3/ZO-3)

  • Verify "no cross-reactivity with other proteins" as indicated in product information

Implementing multiple validation approaches provides confidence in antibody specificity and experimental results.

What are common problems with TJP2 antibody applications and their solutions?

Researchers may encounter several challenges when working with TJP2 antibodies. Here are evidence-based solutions:

Challenge: Weak or No Signal in Western Blot

• Causes: Insufficient protein transfer, epitope masking, antibody degradation

• Solutions:

  • Extend transfer time for high molecular weight TJP2 (150 kDa)

  • Add 0.1% SDS to transfer buffer to facilitate large protein migration

  • Verify antibody activity with positive control lysates (A431, HepG2, U2OS)

Challenge: High Background in Immunohistochemistry

• Causes: Endogenous biotin (for biotin conjugates), insufficient blocking, non-specific binding

• Solutions:

  • Implement avidin/biotin blocking steps for biotin-conjugated antibodies

  • Extend blocking time with 10% serum matching secondary antibody species

  • Increase washing duration and frequency

Challenge: Non-specific Bands in Western Blot

• Causes: Cross-reactivity, protein degradation, non-specific binding

• Solutions:

  • Include protease inhibitors during sample preparation

  • Optimize primary antibody concentration (0.5 μg/mL recommended)

  • Consider alternative blocking agents (BSA vs. milk)

Challenge: Variable Staining Intensity Across Experiments

• Causes: Antibody degradation, inconsistent protocol execution

  • Solutions:

  • Aliquot antibodies to avoid freeze-thaw cycles

  • Standardize incubation times and temperatures

  • Maintain consistent antigen retrieval conditions

These solutions address the most common technical challenges while working with TJP2 antibodies, including biotin-conjugated versions.

How do different fixation methods affect TJP2 antibody performance?

Fixation methods significantly impact TJP2 detection, with important implications for experimental design:

Paraformaldehyde/Formalin Fixation:

• Preserves tissue architecture while maintaining most TJP2 epitopes

• Requires heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

• Optimal for most routine TJP2 detection in tissues

Methanol Fixation:

• Better preserves some TJP2 epitopes but may disrupt membrane structures

• Typically requires less intense antigen retrieval

• Useful for rapid immunofluorescence protocols in cultured cells

Acetone Fixation (for Frozen Sections):

• Provides good epitope preservation with minimal processing

• May offer higher sensitivity but poorer morphological preservation

• Suitable when working with difficult-to-retrieve epitopes

Critical Fixation Parameters:

• Duration: Overfixation (>24 hours) can mask TJP2 epitopes through excessive crosslinking

• Temperature: Room temperature fixation is standard, but cold fixation may better preserve junction structures

• Post-fixation Processing: Careful dehydration and embedding minimizes antigen loss

Understanding these fixation-dependent effects allows researchers to select appropriate protocols based on their specific experimental requirements.

What are the optimal storage conditions for maintaining TJP2 antibody activity?

Proper storage is critical for maintaining TJP2 antibody performance over time:

Storage Recommendations:

Reconstitution Guidelines:

• For lyophilized antibodies, add the recommended volume of distilled water (e.g., 0.2 ml to yield 500 μg/ml)

• Allow complete dissolution before use

• Biotin-conjugated antibodies may benefit from storage in amber tubes to protect from light

Critical Storage Factors:

• Avoid repeated freeze-thaw cycles which significantly reduce antibody activity

• Consider adding carrier protein (BSA) to diluted antibodies for stability

• Storage buffers containing preservatives (e.g., 0.03% Proclin 300) help prevent microbial contamination

Following these storage recommendations ensures consistent antibody performance and experimental reproducibility.

How can TJP2 antibodies contribute to tight junction research?

TJP2 antibodies serve as powerful tools for investigating tight junction biology across multiple research areas:

Barrier Function Studies:

• Quantify TJP2 expression changes during barrier disruption and reformation

• Correlate TJP2 localization with transepithelial/endothelial electrical resistance (TEER) measurements

• Assess junction integrity in disease models affecting barrier function

Junction Assembly and Dynamics:

• Track TJP2 recruitment during de novo junction formation

• Analyze TJP2 redistribution following barrier disruption

• Investigate TJP2 mobility using FRAP (Fluorescence Recovery After Photobleaching) in live cells

Protein-Protein Interaction Analysis:

• Identify TJP2 binding partners through co-immunoprecipitation studies

• Visualize co-localization with other junction components (claudins, occludin)

• Assess cytoskeletal associations critical for junction stability

Pathological Applications:

• Evaluate TJP2 disruption in inflammatory conditions

• Analyze junction remodeling in cancer progression

• Investigate junction integrity in models of blood-brain barrier dysfunction

By employing TJP2 antibodies in these contexts, researchers can gain valuable insights into fundamental aspects of cell-cell adhesion and tissue barrier properties.

What are the considerations for using TJP2 antibodies in clinical research samples?

When applying TJP2 antibodies to clinical research specimens, several special considerations ensure reliable results:

Sample Handling Considerations:

• Minimize cold ischemia time (<1 hour when possible) to preserve junction integrity

• Standardize fixation protocols across all samples for consistent results

• Document fixative type and duration for proper interpretation of TJP2 patterns

Protocol Modifications:

• Optimize antigen retrieval for formalin-fixed, paraffin-embedded (FFPE) clinical samples

• Implement more stringent blocking steps to address variable background in diverse samples

• Consider automated staining platforms for consistent results across large sample sets

Interpretation Challenges:

• Account for variable TJP2 expression across different tissues and pathological states

• Distinguish between membrane-associated and cytoplasmic TJP2 for meaningful analysis

• Implement quantitative image analysis for objective assessment of staining patterns

Controls for Clinical Specimens:

• Include appropriate normal tissue controls alongside pathological samples

• Use serial sections for technical controls (antibody omission, isotype controls)

• Consider multi-site tissue microarrays for standardized evaluation

These considerations enable reliable application of TJP2 antibodies in translational research settings while maintaining scientific rigor.