TJP1 Antibody, HRP conjugated

What is TJP1 and why is it significant in research?

TJP1 (Tight Junction Protein 1), also known as ZO-1, is a membrane-expressed protein that plays critical roles in cellular tight junctions. Research interest in TJP1 has intensified due to its emerging role as a potential therapeutic target and biomarker in cancer research. TJP1 has been identified as significantly involved in the invasion, migration, and proliferation of cancer cells, particularly in lung cancer . The protein has a molecular weight of approximately 195 kDa and functions in maintaining epithelial and endothelial barriers through its scaffolding properties at tight junctions . TJP1's altered expression in various cancer types makes it an important research target for understanding disease progression and developing targeted therapies.

What applications are most suitable for TJP1 Antibody, HRP conjugated?

TJP1 Antibody, HRP conjugated, is particularly valuable for applications requiring direct detection without secondary antibodies. Based on research with non-conjugated versions, the HRP-conjugated format would be especially suitable for:

• Western Blotting (WB): For sensitive protein detection in cell and tissue lysates, allowing for quantification of TJP1 expression levels

• Immunohistochemistry on paraffin-embedded sections (IHC-P): For visualizing TJP1 distribution in tissue samples, particularly in cancer diagnostics research

• ELISA: For quantitative measurement of TJP1 in biological samples

The horseradish peroxidase (HRP) conjugation provides direct enzymatic signal amplification when combined with appropriate substrates, offering advantages in sensitivity and reducing background compared to two-step detection methods in these applications.

How do specificity regions affect antibody selection for TJP1 research?

When selecting a TJP1 antibody for research, the binding specificity region is crucial for experimental success. Different TJP1 antibodies target distinct amino acid sequences within the protein, which can affect recognition of specific isoforms or conformations. For example:

• Antibodies targeting AA 1178-1527 recognize a significant internal portion of human TJP1

• Antibodies targeting AA 1551-1702 bind the C-terminal region

• N-terminal targeting antibodies (N-Term) recognize different structural elements

The choice of specificity region should align with research goals - C-terminal antibodies may better detect full-length protein, while domain-specific antibodies might be necessary when studying protein interactions or when certain epitopes might be masked in experimental conditions. Additionally, human TJP1 shares 82% amino acid sequence identity with mouse TJP1, which is important to consider when planning cross-species experiments .

What are the optimal conditions for Western blotting using TJP1 Antibody, HRP conjugated?

For optimal Western blotting results with TJP1 Antibody, HRP conjugated, researchers should follow these methodological guidelines:

• Sample preparation: Use RIPA buffer with protease inhibitors for efficient extraction of membrane-associated TJP1

• Protein loading: Load 20-40 μg of total protein per lane due to TJP1's high molecular weight (195 kDa)

• Gel selection: Use 6-8% SDS-PAGE gels to properly resolve the large TJP1 protein

• Transfer conditions: Perform overnight transfer at low voltage (30V) at 4°C for efficient transfer of large proteins

• Blocking: Use 5% non-fat dry milk in TBST for 1-2 hours at room temperature

• Antibody dilution: Typically 1:1000-1:5000 dilution in blocking buffer (optimize based on specific antibody concentration)

• Washing: Perform 3-5 stringent washes with TBST to reduce background

• Detection: Use enhanced chemiluminescence (ECL) substrate appropriate for HRP detection

• Exposure time: Start with short exposures (30 seconds) and increase as needed, as HRP-conjugated antibodies often provide strong signals

These optimized conditions are particularly important when studying TJP1 expression differences between normal and cancer tissues, as demonstrated in lung cancer research where differences in expression can be subtle but biologically significant .

How can TJP1 Antibody, HRP conjugated be effectively used in immunohistochemistry?

For successful immunohistochemistry with TJP1 Antibody, HRP conjugated, researchers should implement this methodological approach:

• Fixation: 10% neutral buffered formalin fixation for 24-48 hours

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes

• Endogenous peroxidase blocking: 3% hydrogen peroxide for 10 minutes

• Protein blocking: 5% normal serum for 1 hour

• Primary antibody: Apply TJP1 Antibody, HRP conjugated at 1:50-1:200 dilution and incubate overnight at 4°C

• Substrate development: DAB (3,3'-diaminobenzidine) for 5-10 minutes for visualization of HRP activity

• Counterstaining: Hematoxylin for nuclear visualization

• Scoring system: Implement a standardized scoring system (0 to +++) for expression quantification

This approach has proven effective for examining TJP1 expression patterns across multiple cancer types as shown in comprehensive tissue microarray studies. Research findings demonstrate varied TJP1 expression across cancer types, with particularly notable expression in gastric adenocarcinoma (100%), colon adenocarcinoma (100%), and rectal adenocarcinoma (100%), while lower expression rates were observed in squamous cell carcinomas of the esophagus (33.33%) and skin (0%) .

What controls should be included when using TJP1 Antibody, HRP conjugated?

Rigorous experimental design with appropriate controls is essential when using TJP1 Antibody, HRP conjugated:

Positive controls:

• Cell lines with verified high TJP1 expression (NCI-H69, NCI-H2170 based on relative MFI values of 24.9 and 23.8, respectively)

• Tissues known to express TJP1 (kidney paracancer tissue shows 100% positive staining)

Negative controls:

• Cell lines with minimal TJP1 expression (PC-9, NCI-H23 with relative MFI values of 2.0 and 2.5)

• Tissues with minimal TJP1 expression (liver paracancer tissue shows 0% positive staining)

• Antibody diluent only (no primary antibody) on duplicate sections

Validation controls:

• Knockdown validation using siRNA targeting TJP1 to confirm antibody specificity

• Peptide competition assay using the immunogen peptide (AA 1178-1527) to verify binding specificity

• Western blot correlation to confirm the molecular weight matches the expected 195 kDa band

These controls ensure experimental rigor and support the validity of research findings, particularly when establishing TJP1 as a potential therapeutic target or biomarker.

How does TJP1 expression correlate with cancer progression and prognosis?

TJP1 expression demonstrates significant correlations with cancer progression and patient outcomes, though these relationships vary by cancer type:

Lung Cancer:

• TJP1 knockdown studies demonstrate that reduced expression inhibits invasion, migration, and proliferation of lung cancer cells, suggesting TJP1 plays a promoting role in lung cancer progression

• Antibody microarray screening revealed differential expression of TJP1 in lung cancer tissues compared to normal adjacent tissues

Pancreatic Cancer (PAAD):

• TCGA database analysis revealed TJP1 is highly expressed in pancreatic cancer tissues compared to normal tissues

• Lower TJP1 expression correlates with better prognosis and improved survival rates in PAAD patients

Multiple Cancer Types Expression Profile:
The following table summarizes TJP1 expression across cancer types based on immunohistochemistry:

These expression patterns suggest TJP1 may serve as both a diagnostic marker and therapeutic target, with particular relevance in gastrointestinal and gynecological malignancies .

What methodological approaches are recommended for studying TJP1's role in cancer cell behavior?

To effectively investigate TJP1's role in cancer cell behavior, researchers should implement these methodological approaches:

Gene Expression Manipulation:

• siRNA-mediated knockdown: Transfection of cancer cell lines with TJP1-specific siRNA to reduce expression

• CRISPR-Cas9 gene editing: For complete knockout studies to observe more profound effects

• Overexpression systems: Transfection with TJP1 expression vectors to study gain-of-function effects

Functional Assays:

• Migration assays: Wound healing/scratch assays to assess cell migration capacity

• Invasion assays: Transwell Matrigel invasion assays to assess invasive potential

• Proliferation assays: MTT/CCK-8 assays to measure cell proliferation rates

• Colony formation assays: To assess changes in clonogenic potential

Protein Interaction Studies:

• Co-immunoprecipitation: To identify TJP1-interacting proteins in cancer cells

• Immunofluorescence: To visualize TJP1 localization and co-localization with other proteins

• Proximity ligation assay: For in situ detection of protein interactions

In Vivo Validation:

• Xenograft models: Using TJP1-manipulated cancer cells in immunodeficient mice

• Patient-derived xenografts: To assess TJP1 targeting in more clinically relevant models

• Analysis of patient samples: Correlation of TJP1 expression with clinical parameters

Research has demonstrated that applying these methodologies yields significant insights, as seen in studies where TJP1 knockdown inhibited invasion and migration of lung cancer cells, establishing TJP1 as a potential therapeutic target .

How can researchers interpret conflicting TJP1 expression data across different tumor types?

Interpreting contradictory TJP1 expression data across tumor types requires sophisticated analytical approaches:

Contextual Analysis Framework:

• Tissue-specific biological context: TJP1 functions differently based on tissue origin. For example, expression patterns differ significantly between epithelial cancers (like gastric adenocarcinoma with 100% expression) and non-epithelial tumors (like lymphomas) .

• Subcellular localization differentiation: Membrane-localized versus cytoplasmic TJP1 may have different functional implications. Immunofluorescence studies have confirmed TJP1's membrane expression in lung cancer cells, which correlates with its role in cell migration .

• Quantitative vs. qualitative assessment: Some studies report presence/absence while others report expression levels. The immunohistochemistry scoring system (+, ++, +++) reveals that intensity matters - high-intensity TJP1 staining (+++) is more common in ovarian and prostate cancers than in others .

• Correlation with clinical data: TCGA database analysis showed high TJP1 expression in PAAD correlates with poorer prognosis, while expression patterns in other cancers may not show the same correlation .

• Methodological validation: Cross-validate findings using multiple techniques. The identification of TJP1 involved both antibody microarray screening and confirmation by mass spectrometry with 67% coverage rate of the TJP1 sequence .

• Isoform-specific analysis: Different antibodies target different regions (e.g., AA 1178-1527 vs. AA 1551-1702), which may detect different isoforms with varying functions .

When apparent contradictions emerge, researchers should consider these factors and investigate whether TJP1 might function as either a tumor suppressor or promoter depending on the specific cancer context.

How can TJP1 Antibody, HRP conjugated be used in multiplexed immunoassays?

Multiplexed immunoassays using TJP1 Antibody, HRP conjugated require specialized methodological approaches to maximize information yield while maintaining specificity:

Sequential Multiplex Immunohistochemistry:

• Perform initial staining with TJP1 Antibody, HRP conjugated and develop with one chromogen (e.g., DAB)

• Strip antibodies using glycine buffer (pH 2.2) or commercial antibody stripping solutions

• Block and reprobe with additional antibodies conjugated to different enzymes

• Develop with alternative chromogens (e.g., Vector VIP, AEC)

• Document after each staining round with whole slide scanning

Tyramide Signal Amplification (TSA) Multiplex:

• Apply TJP1 Antibody, HRP conjugated at dilution 1:500-1:1000

• Use fluorophore-conjugated tyramide substrate for signal amplification

• Heat denature antibody while preserving fluorophore signal

• Repeat with additional targets using different fluorophores

• Perform multispectral imaging to separate signals

Spatial Profiling with Digital Pathology:

• Perform multiplexed staining including TJP1 Antibody, HRP conjugated

• Capture high-resolution whole slide images

• Apply digital image analysis algorithms for cell-by-cell quantification

• Correlate with other markers to establish spatial relationships

• Generate tissue maps of TJP1 expression in relation to other markers

These multiplexed approaches enable researchers to simultaneously visualize and quantify TJP1 alongside other markers such as E-cadherin, claudins, and cancer-specific markers, providing deeper insights into TJP1's role in maintaining tissue architecture and its dysregulation in cancer progression .

What strategies can overcome technical challenges when working with TJP1 Antibody, HRP conjugated?

Researchers frequently encounter technical challenges when working with TJP1 Antibody, HRP conjugated. These methodological solutions address common issues:

Challenge: High Background Signal

• Solution: Implement stringent blocking with 3-5% BSA containing 0.1% cold fish skin gelatin

• Rationale: TJP1's membrane localization can lead to nonspecific binding, particularly in tissues with complex architecture

• Validation: Compare background levels between sequential blocking protocols to identify optimal conditions

Challenge: Weak Signal Detection

• Solution: Implement two-layer signal amplification using biotinyl tyramide followed by streptavidin-HRP

• Rationale: Some tissues express lower levels of TJP1 that require amplification despite direct HRP conjugation

• Example: This approach improved detection in liver hepatocellular carcinoma samples where standard methods showed only 33.33% positive staining

Challenge: Epitope Masking in Formalin-Fixed Tissues

• Solution: Extended antigen retrieval (30-40 minutes) with high-pH EDTA buffer (pH 9.0)

• Rationale: The complex secondary structure of TJP1 (195 kDa) requires more aggressive retrieval

• Protocol modification: Implement pressure cooker-based retrieval instead of microwave methods

Challenge: Variability in Expression Quantification

• Solution: Implement standardized digital image analysis with positive cell counting algorithms

• Rationale: Semi-quantitative scoring (+, ++, +++) shows subjective variability between observers

• Standardization: Calibrate against cell lines with known expression levels ranging from high (NCI-H69, MFI: 24.9) to low (PC-9, MFI: 2.0)

Challenge: Cross-Reactivity

• Solution: Validate with peptide competition assays using the immunogenic peptide sequence

• Rationale: Despite claims of "no cross reactivity with other proteins," validation is essential

• Control: Include western blot analysis of knockout/knockdown samples as specificity controls

These methodological approaches significantly improve experimental outcomes when working with TJP1 Antibody, HRP conjugated, particularly in complex tissue environments.

How can researchers design knockdown experiments to study TJP1 function in cancer models?

Designing rigorous TJP1 knockdown experiments requires careful methodological planning to generate reliable and reproducible results:

siRNA Design Strategy:

• Target multiple exons to ensure complete knockdown

• Design 3-4 different siRNA sequences targeting conserved regions of TJP1

• Validate each siRNA individually and create pooled siRNA mixtures to minimize off-target effects

• Include scrambled siRNA controls with similar GC content

Transfection Optimization Protocol:

• Determine cell line-specific transfection conditions using fluorescently-labeled control siRNAs

• Optimize transfection reagent:siRNA ratios (typically 2-3:1 for lipid-based reagents)

• For hard-to-transfect cell lines, employ electroporation or viral vector-based approaches

• Establish transfection timing based on TJP1's protein half-life (typically 48-72 hours post-transfection for functional assays)

Knockdown Validation Requirements:

• Perform qRT-PCR to confirm mRNA reduction (target >70% reduction)

• Validate protein reduction via Western blot using antibodies targeting different epitopes than the knockdown region

• Quantify knockdown efficiency using image analysis software

• Include temporal analysis (24h, 48h, 72h post-transfection) to determine optimal experimental window

Functional Assay Design:

• Migration assays: Wound healing assays at 70-80% confluence post-transfection

• Invasion assays: Transwell Matrigel invasion with 50,000-100,000 cells per insert

• Proliferation assessment: Plate cells 24h post-transfection at 5,000 cells/well in 96-well format

• Include rescue experiments with overexpression of siRNA-resistant TJP1 to confirm specificity

Data Analysis Framework:

• Establish biological replicates (minimum n=3) and technical replicates (minimum n=3)

• Apply appropriate statistical tests (typically ANOVA with post-hoc tests)

• Correlate knockdown efficiency with functional outcomes using regression analysis

• Compare effects across multiple cell lines with varying baseline TJP1 expression levels

This comprehensive approach has demonstrated that TJP1 knockdown inhibits invasion, migration, and proliferation of lung cancer cells, suggesting its potential as a therapeutic target .

How does TJP1 expression analysis contribute to cancer diagnostics and prognostics?

TJP1 expression analysis offers substantial clinical value in cancer diagnostics and prognostics through several methodological approaches:

Diagnostic Applications:

• Differential diagnosis: TJP1 expression patterns differ significantly between cancer types, with gastric, colon, and rectal adenocarcinomas showing consistent positive expression (100%)

• Tissue-of-origin identification: Cancer of unknown primary might be identified through TJP1 expression patterns

• Malignant transformation assessment: Comparison of TJP1 expression between tumor and paracancerous tissues (e.g., kidney clear cell carcinoma shows 33.33% positive staining in tumor vs. 100% in paracancer tissue)

Prognostic Applications:

• Survival correlation: TCGA database analysis revealed that low TJP1 expression correlates with better prognosis in pancreatic adenocarcinoma (PAAD) patients

• Metastatic potential assessment: TJP1's role in cell migration and invasion suggests its utility as a marker for metastatic risk

• Treatment response prediction: Expression levels might predict response to therapies targeting cell adhesion and invasion pathways

Standardized Assessment Protocol:

• Immunohistochemical scoring: Implement standardized scoring systems (0, +, ++, +++) for consistent evaluation

• Digital pathology quantification: Calculate H-scores (intensity × percentage) for more precise quantification

• Multi-marker panels: Integrate TJP1 with other tight junction markers for comprehensive assessment

Clinical Validation Data:

• Pancreatic cancer: TCGA data demonstrates prognostic value of TJP1 expression levels

• Lung cancer: Experimental validation shows functional relevance in migration and invasion, key processes in metastasis

• Cancer type specificity: Expression patterns vary significantly between cancer types, requiring tissue-specific interpretation

This evidence supports TJP1's potential as a clinically relevant biomarker, particularly in gastrointestinal malignancies and pancreatic cancer, where standardized expression analysis could guide treatment decisions and prognostic assessments.

What are the emerging therapeutic strategies targeting TJP1 in cancer?

Emerging therapeutic strategies targeting TJP1 in cancer represent a promising frontier in precision oncology, with several approaches under investigation:

Antibody-Based Therapeutic Approaches:

• Antibody-drug conjugates (ADCs): Conjugating cytotoxic agents to TJP1-targeting antibodies for targeted delivery to cancer cells

• Bispecific antibodies: Engineering antibodies targeting both TJP1 and immune effector cells to enhance immune response against TJP1-expressing tumors

• Antibody-mediated internalization: Utilizing TJP1's potential for internalization to deliver therapeutic payloads intracellularly

Small Molecule Inhibitors:

• Disruption of TJP1 protein-protein interactions: Targeting TJP1's PDZ domains to disrupt essential scaffolding functions

• Destabilizing TJP1 protein: Compounds that accelerate TJP1 degradation

• Allosteric modulators: Compounds that alter TJP1's conformation and impair its function in cancer cells

RNA-Based Therapeutics:

• siRNA delivery systems: Nanoparticle-delivered siRNA to knock down TJP1 expression

• Antisense oligonucleotides: Targeting TJP1 mRNA to inhibit translation

• CRISPR-based approaches: For selective editing of TJP1 in cancer cells

Combination Therapy Strategies:

• Sensitization to conventional chemotherapy: TJP1 inhibition may enhance drug penetration into tumor tissue

• Synergy with anti-angiogenic therapy: Targeting both tumor vasculature and cancer cell invasion/migration pathways

• Immunotherapy combinations: Enhancing immune cell infiltration by disrupting tumor tissue architecture

Preclinical Evidence Supporting These Approaches:

• Knockdown studies demonstrate that reduced TJP1 expression inhibits invasion, migration, and proliferation of lung cancer cells

• TJP1's membrane expression makes it accessible to antibody-based therapeutics

• Differential expression between normal and cancer tissues provides a potential therapeutic window

These therapeutic strategies targeting TJP1 represent promising approaches for cancers where TJP1 is implicated in disease progression, particularly lung cancer and pancreatic adenocarcinoma where expression correlates with poorer prognosis .

What are the emerging techniques for studying TJP1 localization and dynamics?

Advanced methodological approaches for investigating TJP1 localization and dynamics are driving significant research breakthroughs:

Super-Resolution Microscopy Techniques:

• Structured Illumination Microscopy (SIM): Enables visualization of TJP1 within tight junction complexes at ~100nm resolution

• Stochastic Optical Reconstruction Microscopy (STORM): Achieves ~20nm resolution for detailed mapping of TJP1 molecular organization

• Stimulated Emission Depletion (STED) microscopy: Allows live-cell imaging of TJP1 dynamics at subdiffraction resolution

Live-Cell Imaging Approaches:

• CRISPR-mediated endogenous tagging: Knock-in of fluorescent proteins to visualize native TJP1 without overexpression artifacts

• Optogenetic TJP1 manipulation: Light-induced clustering or dissociation of TJP1 complexes to study functional dynamics

• Fluorescence Recovery After Photobleaching (FRAP): Quantification of TJP1 mobility and exchange rates at tight junctions

Proximity-Based Interaction Mapping:

• BioID/TurboID: Proximity-dependent biotinylation to identify transient TJP1 interaction partners

• Split-GFP complementation: Visualization of specific TJP1 protein interactions in living cells

• Förster Resonance Energy Transfer (FRET): Measurement of nanoscale interactions between TJP1 and binding partners

Spatial Multi-omics Integration:

• Spatial transcriptomics combined with TJP1 protein localization

• Multiplexed ion beam imaging (MIBI) for simultaneous detection of dozens of proteins including TJP1

• Correlated light and electron microscopy (CLEM) for ultrastructural context of TJP1 localization

These advanced techniques enable researchers to move beyond static expression analysis to understand the dynamic role of TJP1 in cancer progression, particularly in processes like epithelial-mesenchymal transition and metastatic invasion where tight junction remodeling is critical .

How might single-cell analysis advance our understanding of TJP1's role in tumor heterogeneity?

Single-cell methodologies offer unprecedented insights into TJP1's role in tumor heterogeneity through several innovative approaches:

Single-Cell Protein Analysis:

• Mass cytometry (CyTOF): Quantification of TJP1 protein levels alongside dozens of other markers in individual cells

• Single-cell Western blotting: Direct measurement of TJP1 protein expression and post-translational modifications

• Imaging mass cytometry: Spatial distribution of TJP1 at single-cell resolution within tissue architecture

Single-Cell Transcriptomics:

• scRNA-seq of tumors to identify TJP1 expression variation across cellular subpopulations

• RNA velocity analysis to determine dynamic regulation of TJP1 during cancer cell state transitions

• Trajectory inference to map TJP1 expression changes during tumor evolution

Integrated Multi-modal Analysis:

• CITE-seq: Simultaneous measurement of TJP1 at both protein and mRNA levels in individual cells

• Spatial transcriptomics with protein co-detection: Mapping TJP1 expression patterns within the tumor microenvironment

• Single-cell multi-omics: Correlating TJP1 expression with epigenetic states and proteomic profiles

Functional Single-Cell Assays:

• Micropatterned single-cell migration assays to correlate TJP1 levels with migratory behavior

• Single-cell derived organoids to assess functional consequences of varied TJP1 expression

• CRISPR screens with single-cell readouts to identify genes that modulate TJP1 function

Clinical Applications:

• Patient-derived xenograft models with single-cell resolution analysis of TJP1 expression

• Circulating tumor cell analysis for TJP1 as a marker of metastatic potential

• Prediction of therapy response based on TJP1 expression in resistant subpopulations