TJP1 Antibody, Biotin conjugated
Description
Introduction to TJP1 Antibody, Biotin Conjugated
TJP1 antibody, biotin conjugated, is an immunological reagent consisting of an antibody that specifically recognizes and binds to Tight Junction Protein 1 (also known as Zonula Occludens-1 or ZO-1), which has been chemically modified through the covalent attachment of biotin molecules. This biotinylation process enables the antibody to be detected with exceptional sensitivity through secondary detection systems utilizing avidin or streptavidin conjugates, which exhibit remarkably high affinity for biotin. The resulting detection system provides significant signal amplification compared to conventional labeled antibodies, making it particularly valuable for detecting low-abundance tight junction proteins in complex biological samples .
Biotin conjugated TJP1 antibodies are available in both monoclonal and polyclonal formats, each offering distinct advantages depending on the specific research application. These antibodies can be produced in various host species, with rabbit being the most common, and are typically purified through affinity chromatography to ensure high specificity and minimal background interference .
Target Protein: Structure and Function of TJP1
The target of these specialized antibodies, Tight Junction Protein 1 (TJP1/ZO-1), is a 220-225 kDa peripheral membrane phosphoprotein that belongs to the membrane-associated guanylate kinase (MAGUK) family. TJP1 plays a crucial structural and functional role in cellular tight junctions, which are essential for maintaining epithelial and endothelial barrier integrity throughout the body .
TJP1's molecular structure consists of multiple functional domains that facilitate its diverse cellular roles. These include three PDZ domains at the N-terminus, followed by a SH3 domain and a guanylate kinase-like (GUK) domain. The PDZ domains are particularly important as they mediate interactions with crucial tight junction proteins such as claudins and occludin. The central region of TJP1 contains a sequence that enables interaction with actin filaments, thereby creating a critical connection between the tight junction complex and the cellular cytoskeleton .
Beyond its structural role, TJP1 participates in multiple cellular signaling pathways and developmental processes. It serves as a platform for transmitting signals that regulate cell growth, differentiation, and survival through interactions with transcription factors and regulatory proteins. Research has shown that TJP1 can shuttle between cell junctions and the nucleus, suggesting a role in gene regulation. Additionally, TJP1 participates in the Hippo signaling pathway, which regulates organ size and tissue homeostasis .
Biotin Conjugation Process for TJP1 Antibodies
The production of biotin conjugated TJP1 antibodies involves a specific chemical process known as biotinylation. This procedure typically targets primary amine groups (lysines) on the antibody molecule, although alternative approaches targeting carbohydrate moieties or sulfhydryl groups can also be employed .
The standard solution-phase biotinylation protocol consists of several critical steps:
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Preparation of the antibody by removing sodium azide (which inhibits the reaction) and equilibrating it in an appropriate reaction buffer, typically at a concentration of at least 2 mg/ml for optimal results
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Dissolution of biotin (usually NHS-biotin) in anhydrous dimethyl sulfoxide (DMSO) immediately before use due to its instability
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Addition of dissolved biotin to the antibody solution, typically at a ratio of 80 μg biotin per mg of antibody, with immediate mixing
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Incubation at room temperature for approximately 4 hours, with protection from light
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Removal of unreacted biotin through gel filtration or dialysis, with simultaneous buffer exchange into a suitable storage buffer
For researchers working with limited quantities of antibody, solid-phase biotinylation offers an alternative approach. This method exploits the affinity of IgG-class antibodies for nickel-immobilized metal affinity chromatography (IMAC) supports, enabling on-column derivatization with biotin and minimizing antibody loss during purification steps .
The extent of biotinylation should be carefully controlled, as under-biotinylation reduces detection sensitivity while over-biotinylation can impair antibody binding capacity. Typically, successful conjugation results in 3-6 biotin molecules per antibody molecule, providing optimal balance between detection sensitivity and antigen recognition .
Applications in Research and Diagnostics
Biotin conjugated TJP1 antibodies serve as versatile tools across multiple research and diagnostic applications, with particular utility in techniques requiring high sensitivity and signal amplification. Their primary applications include:
Enzyme-Linked Immunosorbent Assay (ELISA)
In ELISA applications, biotin conjugated TJP1 antibodies typically function as detection antibodies in sandwich assay formats. The standard protocol involves coating the plate with a capture antibody specific to TJP1, followed by sample addition, and then detection using the biotin conjugated TJP1 antibody. Signal generation is accomplished through addition of streptavidin-HRP conjugate and appropriate substrate. This approach enables quantitative detection of TJP1 in various biological samples including serum, plasma, and tissue homogenates, with detection ranges typically spanning 0.156-10 ng/ml and sensitivities below 0.094 ng/ml .
Immunohistochemistry and Immunofluorescence
Biotin conjugated TJP1 antibodies are valuable for visualization of tight junction structures in tissue sections and cell preparations. The biotin-streptavidin detection system provides signal amplification that enhances visualization of both membrane-associated and cyto-nuclear TJP1 distributions. This application has been particularly useful in cancer research, where alterations in TJP1 localization correlate with changes in cellular function and disease progression .
Western Blotting
For protein detection following gel electrophoresis and membrane transfer, biotin conjugated TJP1 antibodies offer enhanced sensitivity compared to conventional detection methods. Typical working dilutions range from 1:300 to 1:5000, with the actual TJP1 protein observed at approximately 195-225 kDa. The biotin-streptavidin detection system reduces background interference while amplifying specific signal .
Flow Cytometry
The high signal-to-noise ratio of biotin-streptavidin systems makes biotin conjugated TJP1 antibodies suitable for flow cytometric applications, particularly when examining cell surface or intracellular TJP1 expression. Working dilutions typically range from 1:20 to 1:100 for this application .
Experimental Protocols for Biotin Conjugated TJP1 Antibodies
The effective utilization of biotin conjugated TJP1 antibodies requires adherence to optimized experimental protocols. The following sections outline key methodologies for major applications:
ELISA Protocol
A standard sandwich ELISA protocol using biotin conjugated TJP1 antibody typically follows these steps:
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Coat microplate wells with anti-TJP1 capture antibody and incubate overnight at 4°C
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Wash and block unbound sites with appropriate buffer
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Add standards or samples and incubate for 90 minutes at 37°C
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Wash thoroughly
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Add biotin-labeled TJP1 antibody working solution (typically diluted 1:100 in antibody dilution buffer) and incubate for 60 minutes at 37°C
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Wash thoroughly
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Add HRP-streptavidin conjugate (SABC) and incubate for 30 minutes at 37°C
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Wash thoroughly
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Add TMB substrate and incubate for 15-30 minutes at 37°C
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Add stop solution and read absorbance at 450 nm
Immunohistochemistry Protocol
For tissue section staining using biotin conjugated TJP1 antibody:
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Deparaffinize and rehydrate sections through xylene and graded alcohols
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Perform antigen retrieval if necessary (typically using citrate buffer pH 6.0)
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Block endogenous peroxidase activity with hydrogen peroxide solution
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Block nonspecific binding with appropriate serum
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Incubate with biotin conjugated TJP1 antibody (typically at 1:200-400 dilution) overnight at 4°C
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Wash thoroughly
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Incubate with streptavidin-HRP conjugate
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Develop with DAB or similar chromogen
Western Blot Protocol
For protein detection using biotin conjugated TJP1 antibody:
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Separate proteins by SDS-PAGE and transfer to appropriate membrane
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Block nonspecific binding with suitable blocking buffer
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Incubate with biotin conjugated TJP1 antibody (typically at 1:2500-5000 dilution) overnight at 4°C
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Wash thoroughly
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Incubate with streptavidin-HRP conjugate
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Wash thoroughly
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Develop using chemiluminescent substrate
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Visualize and analyze bands (TJP1 should appear at approximately 195-225 kDa)
Research Findings Using Biotin Conjugated TJP1 Antibodies
Biotin conjugated TJP1 antibodies have contributed significantly to our understanding of tight junction biology and pathology, particularly in cancer research. Key findings include:
TJP1 in Cancer Progression
Research utilizing biotin conjugated TJP1 antibodies has revealed that changes in TJP1 subcellular localization correlate with cancer progression. Particularly noteworthy is the finding that cyto-nuclear redistribution of TJP1 from membrane-associated tight junctions corresponds with altered inflammatory responses in non-small cell lung cancer. Investigation of TJP1 expression patterns using biotinylated antibodies demonstrated that high cyto-nuclear ZO-1 staining in lung tumor cells was associated with increased density of CD8+ cytotoxic T cells and Foxp3+ immunosuppressive regulatory T cells in the tumor microenvironment .
This research suggests that cyto-nuclear TJP1 influences tumor cell secretome, potentially recruiting immune cells that create a permissive environment for tumor progression. The high sensitivity of biotin-streptavidin detection systems was essential for accurately distinguishing between membrane-associated and cyto-nuclear TJP1 distributions in tissue samples .
TJP1 as a Therapeutic Target in Lung Cancer
High-throughput antibody microarray screening utilizing biotinylated antibodies identified TJP1 as a potential therapeutic target in lung cancer. Immunoprecipitation followed by mass spectrometry confirmed TJP1 as the target of the CL007473 antibody, which showed overexpression in tumor tissue compared to normal lung tissue .
Subsequent functional studies demonstrated that knockdown of TJP1 inhibited invasion, migration, and proliferation of lung cancer cells, suggesting therapeutic potential. This research employed biotin conjugated TJP1 antibodies for both screening and validation phases, highlighting their utility in therapeutic target identification .
Prognostic Significance of TJP1 Expression
Analysis of The Cancer Genome Atlas (TCGA) database combined with immunohistochemical staining using biotin conjugated TJP1 antibodies revealed that TJP1 expression patterns may serve as prognostic indicators in multiple cancer types. Particularly in pancreatic adenocarcinoma (PAAD), lower TJP1 expression correlated with improved patient prognosis and survival .
These findings suggest that biotin conjugated TJP1 antibodies may have diagnostic and prognostic applications beyond their utility as research tools, potentially informing clinical decision-making in oncology.
Comparative Analysis with Other Conjugated Antibodies
The selection of appropriate antibody conjugates for specific applications requires understanding their relative advantages and limitations. Table 2 compares biotin conjugated TJP1 antibodies with other common conjugation types:
| Conjugate Type | Advantages | Limitations | Detection System | Relative Sensitivity |
|---|---|---|---|---|
| Biotin | High signal amplification through avidin/streptavidin; versatile secondary detection options; stable conjugate | Potential background from endogenous biotin; requires secondary detection step | Streptavidin-enzyme or fluorophore | Very high |
| Enzyme direct (HRP/AP) | Single-step detection; no secondary reagent needed | Lower sensitivity than biotin-avidin systems; enzyme stability issues | Direct substrate reaction | Moderate |
| Fluorophore | Direct visualization; suitable for multiplexing; no enzymatic development needed | Photobleaching; lower amplification potential; autofluorescence issues | Direct fluorescence | Moderate |
| Gold | Electron microscopy applications; high stability | Limited signal amplification; size constraints | Direct visualization | Low to moderate |
Research comparing different conjugation methodologies for antibodies indicates that the choice of optimal conjugation depends on the specific application requirements. For enzyme-linked immunosorbent assays (ELISA) and immunohistochemistry applications requiring maximum sensitivity, biotin conjugated antibodies generally outperform direct enzyme conjugates .
A comparative study of conjugation procedures found that the efficiency of biotin conjugation depends on the antibody subclass and isoelectric point, with periodate-mediated conjugations generally providing more efficient conjugates regardless of the antibody subclass when targeting HRP conjugation . For TJP1 antibodies specifically, the biotin conjugation approach should be optimized based on the antibody characteristics and intended application.
Future Perspectives and Emerging Applications
The utility of biotin conjugated TJP1 antibodies continues to expand as new research directions emerge. Several promising areas for future development include:
Multi-omics Integration
The combination of antibody-based detection with mass spectrometry and genomic analyses represents a frontier in tight junction research. Biotin conjugated TJP1 antibodies can serve as pull-down reagents for immunoprecipitation followed by mass spectrometric identification of interaction partners, enabling comprehensive characterization of the tight junction interactome under various physiological and pathological conditions.
Advanced Imaging Applications
Emerging super-resolution microscopy techniques offer unprecedented visualization of subcellular structures. Biotin conjugated TJP1 antibodies, when combined with streptavidin-conjugated quantum dots or other next-generation fluorophores, enable nanoscale visualization of tight junction architecture and dynamic changes during processes such as epithelial-mesenchymal transition.
Therapeutic Development
The identification of TJP1 as a potential therapeutic target in cancer opens avenues for antibody-based therapeutics. Biotin conjugated TJP1 antibodies can facilitate screening and validation of targeted therapeutic approaches, potentially leading to novel treatment strategies for cancers characterized by altered tight junction function.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Dysfunction of the miR-455-TJP1 axis is implicated in bladder cancer cell growth and metastasis. PMID: 30061227
- The tight junction protein ZO-1 exists in stretched and folded conformations within epithelial cells, depending on actomyosin-generated force. PMID: 29199076
- miR103 was upregulated in CRC. Overexpression of miR103 promoted CRC cell proliferation and migration in vitro, whereas downregulation of miR103 inhibited cell proliferation and migration. ZO1 was identified as a direct target of miR103, revealing its expression to be inversely correlated with miR103 expression in CRC samples. PMID: 29115525
- SHANK3 expression correlated with ZO-1 and PKCepsilon in colonic tissue of patients with Crohn's disease. The expression level of SHANK3 affects ZO-1 expression and the barrier function in intestinal epithelial cells. PMID: 28906292
- These results indicate the varying effects of 7-oxygenated cholesterol molecules on the expression and localization of ZO-1 depending on cell types, and suggest the contribution of 7-oxygeneted cholesterol molecules to the structural alteration of tight junctions. PMID: 29428726
- CTR activates AKAP2-anchored cAMP-dependent protein kinase A, which then phosphorylates tight junction proteins ZO-1 and claudin 3. PMID: 28428082
- The Ras signaling pathway is involved in HIV-1 Tat-induced changes in ZO-1 and NEP. PMID: 28553432
- Decreased interaction between ZO-1 and occludin might contribute to the epiphora occurred in the transplanted submandibular glands PMID: 28332063
- Integration of claudin-2, occludin and ZO-1 is necessary for maintaining the function of the proximal tubular epithelium. PMID: 29252987
- Endothelial cellsTLR4 strongly regulates retinal vessel permeability by reducing expression of occludin and zonula occludens 1. PMID: 29136627
- The role of estrogens in the regulation of ZO-1 and estrogen receptors 1 and 2 was evaluated in human primary gut tissues by immunohistochemistry, immunofluorescence and qPCR. PMID: 28867253
- Aberrant expression of the tight junction molecules claudin-1 and zonula occludens-1 mediates cell growth and invasion in oral squamous cell carcinoma cells. PMID: 27436828
- ZO-1-occludin interactions regulate multiple phases of epithelial polarization by providing cell-intrinsic signals that are required for single lumen formation. PMID: 27802160
- It is postulated that ZO-1, when not phosphorylated by PKC, keeps Octn2 in an active state, while elimination of this binding in DeltaPDZ mutant or after ZO-1 phosphorylation leads to diminution of Octn2 activity. PMID: 28257821
- Results uncovered ZO-1 as part of a signaling node activated by VEGF, but not Ang-1, that specifically modulates endothelial cells proliferation during angiogenesis. PMID: 26846344
- Data suggest that long noncoding RNA PlncRNA1 and microRNA miR-34c bound together to regulate the expressions of MAZ, ZO-1 and occludin. PMID: 28153728
- ZO-1 highly expresses in cell-cell junctions and is related to odontoblast differentiation, which may contribute to dental pulp repair or even the formation of an odontoblast layer. PMID: 27109589
- This study shows that the expression and the immunoreactivity of ZO-1 is decreased in the nasal epithelium of patients with allergic rhinitis. PMID: 27216347
- The frequency of alleles and genotypes of rs2291166 gene polymorphism TJP1 was determined in the Mexico Mestizos population. The ancestral allele was the most prevalent. The conformational effect of this amino acid change was performed in silico. PMID: 26259745
- We identified potential nuclear and membrane biomarkers (increased expression of ZO-1, caveolin-1 and P2X7 receptor) of risks for placenta and pregnancy. PMID: 26657896
- OCLN and ZO1 levels appear to be early prognostic markers in patients suffering from sepsis. PMID: 26863122
- The data suggest that ZO-1, along with CD38 and Zap-70, plays a role in cell cycle regulation in chronic B cell leukemia, and may be used as a prognostic marker in the disease monitoring. PMID: 26306999
- These data provide the first evidence that beta-catenin and ZO-1 are direct targets of E7 of the oncogenic beta-human papillomavirus types 5 and 8. PMID: 26645068
- Report TNF-alpha/Il6 mediated dysregulation of zonula occludens-1 properties in human brain microvascular endothelium. PMID: 25953589
- Upon specific knockdown of the accessory TJP, ZO-1, undifferentiated NSCs showed decreased levels of key stem cell markers. PMID: 25892136
- HTT may inhibit breast tumor dissemination through maintenance of ZO1 at tight junctions. PMID: 26293574
- These results suggest that the localization of ZO-1 in cell-cell contacts is differently regulated by activation and inhibition of JNK and/or p38 MAPK depending on the incubation period. PMID: 25435485
- CFTR colocalizes with ZO-1 at the tight junctions of trachea and epididymis, and is expressed before ZO-1 in Wolffian ducts. PMID: 25107366
- Phosphorylation state of the tyrosine of claudin-1 and claudin-2 regulates interaction with ZO1. PMID: 26023235
- miR-18a and RUNX1 could reversely regulate the permeability of blood-tumor barrier as well as the expressions and distributions of ZO-1, occludin and claudin-5. PMID: 25452107
- Zonula occludens-1, occludin and E-cadherin expression and organization in salivary glands. PMID: 25248927
- ZO-1 was internalized and shown to accumulate in the cytoplasm of human podocytes in an IL-13 dose-dependent manner. PMID: 25683991
- The decreased UCP2 expression and increased ZO-1 expression suggest that the oxidative stress-induced mitochondrial dysfunction and tight junction formation may play pivotal roles in the progress of NVG. PMID: 23835672
- ZO-1 is a central regulator of VE-cadherin-dependent endothelial junctions that orchestrates the spatial actomyosin organization. PMID: 25753039
- Tjp1 expression was decreased in glomerular diseases in human and animal models, our results indicate that the suppression of Tjp1 could directly aggravate glomerular disorders, highlighting Tjp1 as a potential therapeutic target. PMID: 25184792
- In conclusion, our present study indicated that miR-34c regulated the permeability of BTB via MAZ-mediated expression changes of ZO-1, occludin, and claudin-5. PMID: 25201524
- ZO-1 showed a tendency to be detected more intensely in myocardial infarction and ischemic heart disease myocardial tissue then in asphyxiation or drowning. PMID: 24368520
- ZO-1 gene shows a hypermethylation status in children with NHL. PMID: 24927439
- Gene expression is regulated by p38MAPK. PMID: 23856837
- High expression of ZO-1 is associated with good prognosis in non-small cell lung cancer. PMID: 24294375
- The LIM domain protein FHL1C interacts with tight junction protein ZO-1 contributing to the epithelial-mesenchymal transition of a breast adenocarcinoma cell line. PMID: 24657059
- Luciferase assays and chromatin immunoprecipitation assays showed that KLF4 up-regulated the promoter activities and interacted with "CACCC" DNA sequence presented in the promoters of ZO-1, occludin, and claudin-5. PMID: 24318462
- Our results identify a novel regulatory pathway involving the interplay between ZO-1, alpha5-integrin and PKCepsilon in the late stages of mammalian cell division. PMID: 23967087
- Data suggest components of diet supplements (here, glutamine/arginine) can improve permeability and tight junction protein expression (TJP1/occludin) in enterocytes exposed to deleterious effects of antineoplastic agents (here, methotrexate). PMID: 23428392
- ZO-1 expression is correlated with malignant phenotypes of GIST. PMID: 23820955
- Vascular endothelial tight junctions and barrier function are disrupted by 15(S)-hydroxyeicosatetraenoic acid partly via protein kinase C epsilon-mediated zona occludens-1 phosphorylation at threonine 770/772. PMID: 24338688
- The presence of neural cells (PC12 cells or trigeminal neurons) markedly promoted the stratification of HCE cells as well as increased the amounts of N-cadherin mRNA and protein in these cells. PMID: 24327615
- The methylation positivity rates of the ID4 and ZO-1 genes in the bone marrow and paraffin-embedded lymphoma tissues of non-Hodgkin lymphoma patients were significantly higher compared to the rates in the Hodgkin lymphoma patients. PMID: 23670122
- Proteomic identification of ZO-1 binding partners and associated proteins that form tight junction complexes. PMID: 23553632
- Single nucleotide polymorphisms in TJP1 is associated with response to antipsychotic agents in schizophrenia. PMID: 23241943
Q&A
What is TJP1 and why is it an important research target?
TJP1 (Tight Junction Protein 1), also known as ZO-1, is a membrane-expressed protein that plays a crucial role in cellular tight junctions. It has emerged as an important research target due to its involvement in various cellular processes including cell adhesion, barrier function, and signal transduction. Recent studies have identified TJP1 as a potential therapeutic target for lung cancer, where its expression correlates with cancer cell invasion, migration, and proliferation capabilities . TJP1 expression has also been linked to prognostic outcomes in pancreatic cancer and other malignancies, making it a valuable biomarker for cancer research . Understanding TJP1's function and expression patterns can provide insights into both normal physiology and disease pathogenesis.
How does a biotin-conjugated TJP1 antibody differ from unconjugated variants?
Biotin-conjugated TJP1 antibodies contain biotin molecules covalently attached to the antibody structure, unlike their unconjugated counterparts described in the search results . This biotinylation offers significant advantages in research applications due to the extremely high affinity between biotin and streptavidin (or avidin). This strong interaction enables enhanced signal amplification in detection systems when used with streptavidin-conjugated reporter molecules (e.g., streptavidin-HRP, streptavidin-fluorophores). The biotinylation process preserves the antibody's binding specificity while providing greater flexibility in detection strategies. Researchers should note that while biotinylation enhances detection sensitivity, it may occasionally affect the antibody's ability to recognize certain epitopes if the biotin molecules are conjugated near the antigen-binding site.
What epitopes of TJP1 are typically targeted by commercially available antibodies?
Commercial TJP1 antibodies typically target specific amino acid sequences within the protein. Based on available data, antibodies commonly target regions such as amino acids 1178-1527 , 1551-1702 , or internal portions of the human ZO-1 protein . Different epitope targets may be suitable for different applications and species. For instance, antibodies targeting AA 1178-1527 show reactivity with human, mouse, and rat samples . When selecting a biotin-conjugated TJP1 antibody, researchers should carefully consider the specific epitope recognition in relation to their experimental design and target species, as epitope accessibility may vary depending on protein conformation and experimental conditions.
What are the optimal applications for biotin-conjugated TJP1 antibodies?
Biotin-conjugated TJP1 antibodies excel in applications requiring signal amplification or multi-layered detection systems. Based on the applications of unconjugated variants, biotin-conjugated TJP1 antibodies would be particularly valuable for:
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Immunohistochemistry: Especially in tissues with low TJP1 expression levels
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Immunofluorescence: For high-sensitivity detection of membrane-localized TJP1
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Flow cytometry: For quantitative analysis of TJP1 expression across different cell populations
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Multiplex assays: Where detection of multiple targets simultaneously benefits from the flexible biotin-streptavidin detection system
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ELISA: Where signal amplification improves sensitivity for detecting low concentrations of TJP1
The biotin-streptavidin system provides greater sensitivity compared to direct detection methods, making it ideal for detecting limited quantities of TJP1 in research samples.
How should researchers optimize immunofluorescence protocols when using biotin-conjugated TJP1 antibodies?
When performing immunofluorescence with biotin-conjugated TJP1 antibodies, researchers should consider the following methodological optimizations:
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Blocking step: Include an avidin/biotin blocking step to reduce endogenous biotin-related background, particularly in tissues with high endogenous biotin (e.g., liver, kidney)
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Antibody concentration: Typically requires lower concentrations (approximately 0.5-2 μg/mL) than unconjugated antibodies due to signal amplification
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Secondary detection: Use streptavidin conjugated to appropriate fluorophores (e.g., Alexa Fluor 488, 594, 647) based on experimental design
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Membrane permeabilization: Since TJP1 is primarily membrane-expressed , gentle permeabilization (0.1-0.2% Triton X-100 for 5-10 minutes) helps maintain membrane integrity while allowing antibody access
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Controls: Include both positive controls (tissues known to express TJP1) and negative controls (isotype control or pre-adsorption with immunizing peptide)
Research demonstrates that TJP1 shows primarily membrane localization as verified by immunofluorescence assays , which can serve as a quality control check for antibody specificity and protocol optimization.
What strategies help resolve contradictory TJP1 expression data between different detection methods?
When facing contradictory TJP1 expression data between different methods (e.g., Western blot vs. immunofluorescence), researchers should systematically address potential sources of discrepancy:
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Epitope accessibility: Different applications expose different protein epitopes. For membrane proteins like TJP1, native conformation (preserved in immunofluorescence) may differ from denatured form (in Western blotting)
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Antibody validation: Verify antibody specificity using knockdown/knockout controls as demonstrated in TJP1 research where siRNA knockdown confirmed antibody specificity
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Signal quantification: For biotin-conjugated antibodies, establish standard curves with recombinant TJP1 protein to assess signal linearity across detection methods
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Cross-reactivity analysis: Test against related junction proteins to ensure specificity as some antibodies show "no cross reactivity with other proteins"
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Method-specific controls: Include cell lines with known differential TJP1 expression (e.g., NCI-2170 and SK-LU-1 with high expression versus 1G2 and PC9 with low expression)
A comparative analysis approach combining multiple detection methods provides the most reliable assessment of true TJP1 expression patterns.
How can biotin-conjugated TJP1 antibodies be utilized in cancer research?
Recent research has established TJP1 as a potential therapeutic target and prognostic marker in cancer . Biotin-conjugated TJP1 antibodies can enhance cancer research through:
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Tumor tissue microarray analysis: High-throughput screening of TJP1 expression across multiple patient samples with enhanced sensitivity
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Circulating tumor cell detection: Using flow cytometry with biotin-conjugated TJP1 antibodies to identify cancer cells with altered tight junction protein expression
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Prognostic marker evaluation: Quantitative assessment of TJP1 expression in relation to patient outcomes, particularly in lung and pancreatic cancer
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Drug response monitoring: Evaluating changes in TJP1 expression following treatment with targeted therapies
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Cancer subtype classification: Correlating TJP1 expression patterns with specific cancer subtypes, such as lung adenocarcinoma versus squamous cell carcinoma
Research data indicates that knockdown of TJP1 in lung cancer cell lines inhibits invasion, migration, and proliferation, suggesting that accurate quantification of TJP1 using sensitive detection methods is valuable for understanding cancer progression mechanisms .
What are the critical factors to consider when using biotin-conjugated TJP1 antibodies in multi-color flow cytometry?
When incorporating biotin-conjugated TJP1 antibodies into multi-color flow cytometry panels, researchers should address these technical considerations:
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Panel design: Select streptavidin-conjugated fluorophores that minimize spectral overlap with other fluorophores in your panel
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Titration: Determine optimal antibody concentration using a titration series (typically 0.1-5 μg/mL) to achieve maximum signal-to-noise ratio
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Compensation controls: Include single-stained controls for each fluorophore, including the streptavidin-fluorophore conjugate
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Blocking strategy: Implement comprehensive blocking of Fc receptors and endogenous biotin to reduce non-specific binding
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Gating strategy: Establish consistent gating based on known TJP1 expression patterns in control cell lines, such as the differential expression observed in lung cancer cell lines (e.g., high expression in NCI-2170 and SK-LU-1 with relative mean fluorescent intensity (MFI) of 23.8 and 14.6, versus low expression in 1G2 and PC9 cell lines with relative MFI of 2.5 and 2.0)
This approach enables reliable quantification of TJP1 expression across heterogeneous cell populations with enhanced sensitivity.
How do post-translational modifications of TJP1 affect antibody recognition?
Post-translational modifications (PTMs) of TJP1 can significantly impact antibody recognition, particularly with biotin-conjugated antibodies:
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Phosphorylation: TJP1 undergoes phosphorylation at multiple sites which can alter protein conformation and epitope accessibility
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Ubiquitination: Affects protein stability and may mask epitopes recognized by specific antibodies
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Palmitoylation: Critical for membrane association of TJP1 and may influence detection of membrane-expressed TJP1
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Epitope-specific considerations: Antibodies targeting different regions (e.g., AA 1178-1527 versus AA 1551-1702 ) may exhibit differential sensitivity to specific PTMs
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Experimental validation: Researchers should validate biotin-conjugated antibodies against samples with altered PTM profiles (e.g., phosphatase-treated versus untreated samples)
Understanding these interactions is crucial when studying regulatory mechanisms affecting TJP1 function in tight junction assembly and stability.
What are the most common causes of false-positive or false-negative results when using biotin-conjugated TJP1 antibodies?
Researchers commonly encounter the following issues that lead to misleading results:
False-positive results:
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Endogenous biotin: Particularly problematic in biotin-rich tissues (kidney, liver) where inadequate blocking causes high background
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Cross-reactivity: Some antibodies may recognize proteins with similar epitopes, though high-quality antibodies show "no cross reactivity with other proteins"
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Non-specific binding: Inadequate blocking or excessive antibody concentration can increase background signal
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Detector saturation: Over-amplification due to extended incubation with streptavidin conjugates
False-negative results:
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Epitope masking: Certain fixation methods may alter the TJP1 epitope, reducing antibody recognition
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Insufficient permeabilization: As a membrane protein , TJP1 detection requires appropriate membrane permeabilization
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Sample degradation: Proteolytic degradation of TJP1 during sample preparation
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Biotin-streptavidin interference: Excess free biotin in samples can compete with biotinylated antibodies
Implementing appropriate controls and validation steps ensures reliable interpretation of TJP1 detection results.
How can researchers validate the specificity of biotin-conjugated TJP1 antibodies?
Comprehensive validation of biotin-conjugated TJP1 antibodies should include:
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Knockdown/knockout controls: siRNA knockdown of TJP1 (as demonstrated in NCI-2170 and SK-LU-1 cells with siRNA-5274) should produce corresponding reduction in antibody signal
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Pre-adsorption controls: Pre-incubation of antibody with immunizing peptide should eliminate specific staining
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Multiple antibody comparison: Use antibodies targeting different TJP1 epitopes (e.g., AA 1178-1527 , AA 1551-1702 ) to confirm staining patterns
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Comparison with unconjugated version: Signal pattern should be consistent between biotin-conjugated and unconjugated versions of the same antibody
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Cross-species validation: If the antibody claims cross-reactivity with multiple species (e.g., human, mouse, rat) , confirm consistent staining patterns across species with known TJP1 expression
How are biotin-conjugated TJP1 antibodies contributing to our understanding of tight junction dynamics in disease?
Biotinylated TJP1 antibodies are enabling advanced research into tight junction dynamics through:
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Live-cell imaging: Using cell-permeable biotin-conjugated antibody fragments to track TJP1 mobilization during junction assembly and disassembly
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Super-resolution microscopy: Enhanced visualization of TJP1 clustering and interaction with other junction proteins
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Cancer research applications: Investigation of TJP1's role in regulating invasion and migration of cancer cells, as demonstrated in lung cancer studies
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Quantitative assessment: Precise measurement of TJP1 expression levels across different cancer types and correlation with patient outcomes, especially in pancreatic cancer
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Drug development: Screening compounds that modulate TJP1 expression or function as potential therapeutic strategies
These applications are particularly valuable as research has demonstrated TJP1's involvement in cancer cell function, with knockdown studies showing reduced invasion, migration, and proliferation in lung cancer cell lines .
What recent technological advances are improving the utility of biotin-conjugated antibodies in TJP1 research?
Recent technological developments enhancing biotin-conjugated TJP1 antibody applications include:
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Site-specific biotinylation: Precisely controlling biotin location on the antibody to avoid interference with antigen binding
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Multiplexed detection systems: Combining biotin-streptavidin interactions with other detection chemistries for simultaneous analysis of multiple targets
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Microfluidic platforms: Integration of biotinylated antibodies into lab-on-chip devices for high-throughput screening of TJP1 expression
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Mass cytometry (CyTOF): Utilizing metal-tagged streptavidin for highly multiplexed analysis of TJP1 in relation to dozens of other cellular markers
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Antibody engineering: Development of recombinant antibody fragments with optimized biotinylation sites for improved sensitivity and specificity
These advances are particularly relevant for cancer research applications where precise quantification of TJP1 expression can provide insights into disease progression and treatment response .
