clr6 Antibody

What are Claudin-6 and CXCR6 antibodies and what cellular structures do they target?

Claudin-6 antibodies target the Claudin-6 protein (CLDN6), a member of the claudin family of tight junction proteins. This protein spans from Met1-Val220 and is encoded by the gene with accession number P56747 . Claudin-6 is primarily localized to cell surfaces and plays a critical role in maintaining epithelial cell barriers.

CXCR6 antibodies target the CXCR6 chemokine receptor protein (spanning Met1-Leu342, accession number O00574), which functions as a receptor for the transmembrane chemokine CXCL16 . CXCR6 is expressed on various immune cells, including T cells and natural killer cells, and is involved in chemotaxis and migration processes.

Both antibodies are valuable tools for studying cellular localization, protein expression, and functional interactions in their respective biological pathways.

What cell and tissue types show detectable expression of Claudin-6 and CXCR6?

Claudin-6 expression has been detected in:

• Human induced pluripotent stem cells (iPSCs), particularly when differentiated into definitive endoderm

• Human embryonic tissues

• Certain cancer cell types, including hepatocellular carcinoma as indicated by studies examining its potential as an oncofetal target

CXCR6 expression has been observed in:

• Human peripheral blood mononuclear cells (PBMCs), with expression enhanced after IL-2 treatment

• T lymphocytes, particularly those expressing CD3e

• Various cancer tissues, including lung cancer, pancreatic ductal adenocarcinoma, and melanoma

• Polymorphonuclear neutrophils in pancreatic carcinoma and acute bacterial infections

The expression profile of both proteins makes them important markers in developmental biology, cancer research, and immunology.

What are the established experimental applications for these antibodies?

Both antibodies have been validated for multiple experimental applications:

Claudin-6 Antibody Applications:

• Flow cytometry for detecting expression in transfected cells

• Immunocytochemistry/Immunofluorescence (ICC/IF) for visualizing cellular localization

• Studies of definitive endoderm differentiation from pluripotent stem cells

CXCR6 Antibody Applications:

• Flow cytometry for immune cell population analysis

• Immunohistochemistry (IHC) for tissue expression analysis

• Immunocytochemistry for cellular localization studies

• Western blotting (for some tissue types)

These applications enable researchers to investigate protein expression, localization, and function across various experimental systems.

How does antibody specificity against membrane proteins like Claudin-6 and CXCR6 impact experimental design?

Membrane protein antibody specificity requires careful experimental design considerations. For Claudin-6, specificity has been shown to be driven by single atomic contact points, highlighting the precision required in antibody-antigen interactions . This molecular specificity has significant implications:

• Proper controls must include both isotype controls and irrelevant protein transfection controls, as demonstrated in the validation studies for both antibodies

• When investigating highly conserved membrane proteins, researchers should validate antibody specificity using transfected cell lines expressing the target protein alongside control transfectants

• Cross-reactivity testing is essential, particularly when studying proteins with high sequence homology to other family members

For membrane proteins, permeabilization protocols must be carefully optimized to preserve epitope structure while allowing antibody access. This is particularly important for multi-pass membrane proteins like Claudin-6 and CXCR6.

What role do these proteins and their corresponding antibodies play in understanding disease mechanisms?

Research using these antibodies has contributed significantly to understanding disease mechanisms:

Claudin-6 in Disease:

• Identified as a potential oncofetal target in hepatocellular carcinoma

• Associated with pluripotency and differentiation pathways

• Implicated in hepatitis C virus infection through claudin specificity interactions

CXCR6 in Disease:

• Overexpressed in various cancers including lung cancer, with coexpression of its ligand CXCL16 associated with invasion capacity

• Involved in the LILRB1-HLA-G axis defining checkpoints in natural killer cell exhaustion in tuberculosis

• Expressed in pancreatic ductal adenocarcinoma and potentially involved in disease progression

• Implicated in autoimmune conditions including systemic lupus erythematosus

• Shown to mediate reverse signaling via CXCL16, suggesting complex signaling networks

These findings demonstrate how antibodies against these targets serve as crucial tools for investigating complex disease mechanisms and potential therapeutic approaches.

How can researchers effectively use these antibodies in multi-parameter analyses?

Multi-parameter analyses require careful planning and optimization:

• Panel design considerations:

  • Both antibodies have been successfully used in multiplexed assays, as demonstrated by dual staining of Claudin-6 with SOX17 and CXCR6 with CD3e

  • When designing panels, select fluorophores with minimal spectral overlap and appropriate brightness for expected expression levels

• Compensation requirements:

  • Proper compensation controls should be established using single-stained samples

  • For flow cytometry applications, follow established protocols for staining membrane-associated proteins as referenced in the data sheets

• Integration with other techniques:

  • Consider complementary approaches such as CyTOF (mass cytometry) for higher parameter analyses, as demonstrated in the study of natural killer cell exhaustion in tuberculosis

  • Spatial information can be preserved by combining immunofluorescence with other techniques like in situ hybridization

What are the optimal protocols for immunostaining with these antibodies?

For Claudin-6 Immunofluorescence:

• Concentration: 10 μg/mL has been validated

• Incubation time: 3 hours at room temperature

• Detection system: Compatible with secondary antibodies such as NorthernLights 557-conjugated Anti-Mouse IgG

• Counterstaining: DAPI for nuclear visualization

• Special considerations: Works effectively in immersion-fixed iPSC cells differentiated into definitive endoderm

For CXCR6 Immunohistochemistry/Immunofluorescence:

• Both paraffin-embedded (IHC-P) and frozen sections are compatible

• Detection systems: Both chromogenic (DAB) and fluorescent detection methods have been validated

• Quantification: Immuno-intensities can be quantified using image analysis software (e.g., Aperio ImageScope)

• Background reduction: Include appropriate blocking steps to minimize non-specific binding

Both antibodies require optimization for specific tissue types and fixation methods, with preliminary titration experiments recommended to determine optimal concentration for each application.

How should researchers approach validation of these antibodies in their experimental systems?

A comprehensive validation approach should include:

• Positive and negative controls:

  • Positive controls: For Claudin-6, use transfected HEK293 cells or differentiated iPSCs ; for CXCR6, use IL-2 treated PBMCs or relevant cancer cell lines

  • Negative controls: Include isotype controls (e.g., Mouse IgG2B for Claudin-6), irrelevant protein transfectants, and tissues known not to express the target protein

• Expression correlation:

  • Correlate protein detection with mRNA expression where possible

  • Consider using multiple antibody clones targeting different epitopes

• Functional validation:

  • For CXCR6, validation can include functional assays such as chemotaxis or signaling responses to CXCL16

  • For Claudin-6, barrier function assays or interaction studies may provide functional validation

• Knockdown/knockout verification:

  • Antibody staining should be reduced or absent in cells with genetic deletion or knockdown of the target protein

What considerations should guide antibody selection for specific disease models?

When selecting antibodies for disease models, researchers should consider:

• Expression patterns relevant to the disease:

  • CXCR6 antibodies are particularly valuable in studying lung cancer, where the protein is coexpressed with its ligand CXCL16 and mediates invasion

  • Claudin-6 antibodies may be more appropriate for developmental studies or hepatocellular carcinoma research

• Application-specific performance:

  • For flow cytometry of immune cells, the CXCR6 antibody has been extensively validated in multiple studies of various immune subpopulations

  • For developmental biology studies, the Claudin-6 antibody has demonstrated utility in stem cell differentiation models

• Species compatibility:

  • While both antibodies target human proteins, cross-reactivity with other species should be confirmed if working with animal models

• Disease-specific modifications:

  • Consider whether post-translational modifications or splice variants in disease states might affect antibody binding

What are common issues encountered when using these antibodies in flow cytometry?

Common flow cytometry challenges and solutions include:

• Low signal intensity:

  • Increase antibody concentration (within recommended ranges)

  • Optimize staining conditions (time, temperature, buffer composition)

  • Ensure cells are properly prepared with minimal cell death

  • For CXCR6, consider IL-2 pretreatment to upregulate expression in PBMCs as demonstrated in validation studies

• High background:

  • Improve blocking steps (use both Fc block and protein-based blockers)

  • Optimize washing steps (increase number or volume of washes)

  • Ensure proper compensation when using multiple fluorochromes

  • Set quadrant markers based on appropriate isotype controls as demonstrated in the protocols

• Variable expression:

  • Standardize cell preparation methods

  • Consider expression kinetics (e.g., CXCR6 expression changes with cytokine stimulation)

  • Account for heterogeneity in primary samples

• Membrane protein staining challenges:

  • Use protocols specifically designed for membrane proteins

  • Consider live-cell staining for surface epitopes

  • Avoid harsh fixation that may distort membrane protein epitopes

How can researchers address reproducibility challenges in antibody-based experiments?

To improve reproducibility:

• Standardize protocols:

  • Document detailed protocols including antibody concentration, incubation time, buffer composition, and washing steps

  • Maintain consistent cell/tissue preparation methods between experiments

• Antibody validation and quality control:

  • Use antibodies with comprehensive validation data like those shown in the search results

  • Record lot numbers and perform lot-to-lot validation when necessary

  • Consider preparing large batches of working dilutions to minimize variability

• Quantitative approaches:

  • Use quantitative image analysis for IHC/ICC as demonstrated in the CXCR6 lung cancer studies

  • Apply standardized gating strategies for flow cytometry

  • Include calibration standards where possible

• Biological variability management:

  • Increase biological replicates to account for natural variation

  • Include consistent positive and negative controls in each experiment

  • Consider the impact of factors like cell density, passage number, and donor variability

What strategies exist for optimizing signal-to-noise ratio in immunofluorescence applications?

To optimize signal-to-noise ratio:

• Fluorophore selection:

  • Choose fluorophores with spectral properties suited to your microscopy setup

  • Consider signal amplification systems for low-abundance targets

  • The Claudin-6 detection protocol using NorthernLights 557-conjugated secondary antibody represents a validated approach

• Sample preparation optimization:

  • Optimize fixation to preserve antigen while maintaining tissue morphology

  • Test different permeabilization conditions if intracellular epitopes are targeted

  • Consider antigen retrieval methods when working with formalin-fixed tissues

• Background reduction:

  • Implement thorough blocking steps (serum from secondary antibody species)

  • Use detergents at appropriate concentrations to reduce non-specific binding

  • Include autofluorescence quenching steps, particularly for tissues with high endogenous fluorescence

• Image acquisition and processing:

  • Optimize exposure settings to prevent saturation

  • Use appropriate filters to minimize bleed-through

  • Consider deconvolution or other post-processing methods to enhance signal clarity

How are these antibodies contributing to cancer research and potential therapeutic approaches?

Both antibodies have demonstrated significant utility in cancer research:

Claudin-6 in Cancer Research:

• Identified as a potential oncofetal target in hepatocellular carcinoma, suggesting applications in both diagnostic and therapeutic approaches

• The high specificity of the antibody, driven by specific atomic contact points, makes it valuable for targeted therapy development

• Studies exploring viral infection mechanisms mediated by claudins may inform approaches to prevent cancer-associated viral infections

CXCR6 in Cancer Research:

• Extensively studied in lung cancer, where CXCR6/CXCL16 coexpression correlates with invasion capacity

• CXCR6 expression has been quantitatively analyzed in lung adenocarcinoma and squamous cell carcinoma specimens, showing significant upregulation compared to non-neoplastic tissues

• Implicated in melanoma plasticity through microRNA-222 regulation

• Associated with signaling pathways in gastric cancer progression through STAT3-mediated expression of Ror1

• Demonstrated role in pancreatic ductal adenocarcinoma progression

• Possible target for immunomodulatory approaches in cancer, given its role in immune cell trafficking

These findings highlight how antibodies against these targets contribute to understanding cancer mechanisms and identifying potential therapeutic targets.

What insights do these antibodies provide into stem cell biology and development?

These antibodies have contributed valuable insights into developmental processes:

Claudin-6 in Development:

• Successfully used to detect Claudin-6 in human induced pluripotent stem cells differentiated into definitive endoderm, providing markers for tracking differentiation

• Co-localization studies with developmental transcription factors like SOX17 help elucidate the relationship between tight junction formation and lineage commitment

CXCR6 in Stem Cell Biology:

• Studies using the CXCR6 antibody have examined the migration capacity of mesenchymal stem cells, correlating receptor expression with migratory potential

• Research has explored the relationship between CXCR6 expression and homing behavior of regulatory T cells in response to vitamin A metabolites

These applications demonstrate how these antibodies serve as valuable tools for tracking developmental processes and stem cell behavior.