CLDND1 Antibody

What is CLDND1 and what cellular functions does it perform?

CLDND1 (Claudin domain-containing protein 1) is a member of the tight junction protein family, also known by several aliases including C3orf4, HSPC174, PSEC0054, UNQ2511/PRO6000, and GENX-3745 . It is a small protein (approximately 29 kDa, 253 amino acids) with four transmembrane domains, with both N-terminus and C-terminus located in the cytoplasm .

CLDND1 functions primarily as a component of tight junctions, where it helps establish paracellular barriers that control molecular flow in the intercellular space between epithelial cells . While its role when incorporated into tight junctions is well-established, the function of nonjunctional CLDN1 (njCLDN1) is less understood but appears significant in processes like tissue fibrosis .

What types of CLDND1 antibodies are available for research applications?

CLDND1 antibodies are available in various formats to suit different experimental needs. The primary types include:

Most commercially available CLDND1 antibodies are rabbit polyclonal antibodies that react with human, mouse, and rat samples . These antibodies are typically generated using CLDND1 fusion proteins as immunogens and purified through antigen affinity purification . For specialized research, monoclonal antibodies targeting specific epitopes, such as the 6F6 mAb against CLDND1's extracellular domain, have been developed for potential therapeutic applications .

What are the optimal methods for validating CLDND1 antibody specificity?

Validating CLDND1 antibody specificity requires multiple complementary approaches:

• Western blotting with positive and negative controls: Confirm the antibody detects a band at the expected molecular weight (29 kDa) in tissues known to express CLDND1 while showing minimal reactivity in tissues with low expression .

• Immunohistochemistry with proper controls: Compare staining patterns in tissues with known CLDND1 expression profiles. For instance, in normal tissues, CLDND1 is predominantly cytoplasmic, while in colorectal cancer samples, it shows primarily membranous localization .

• Knockdown/knockout validation: Use siRNA or CRISPR techniques to reduce CLDND1 expression in cell lines, then confirm decreased signal with the antibody.

• Cross-reactivity testing: Evaluate potential cross-reactivity with other claudin family members, particularly in experiments involving tissues expressing multiple claudin proteins.

• Comparison of multiple antibodies: When possible, use antibodies targeting different epitopes of CLDND1 to confirm consistent staining patterns.

What are the optimal conditions for using CLDND1 antibodies in immunohistochemistry?

For optimal immunohistochemical detection of CLDND1:

When interpreting results, note that CLDND1 localization differs between normal and pathological tissues - it's predominantly cytoplasmic in normal tissues but membrane-localized in some cancers like CRC .

How should researchers optimize Western blot protocols for CLDND1 detection?

For successful Western blot detection of CLDND1:

• Sample preparation: Use RIPA buffer supplemented with protease inhibitors. For membrane proteins like CLDND1, addition of 0.1% SDS can improve extraction.

• Protein loading: Load 20-50 μg of total protein per lane.

• Gel conditions: Use 12-15% SDS-PAGE gels to adequately resolve the 29 kDa CLDND1 protein .

• Transfer conditions: Semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour to PVDF membranes.

• Blocking: 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Antibody incubation: Dilute primary antibody (typically 1:500-1:2000) in blocking buffer and incubate overnight at 4°C. Use HRP-conjugated secondary antibodies.

• Washing: Thorough washing with TBST (at least 3 × 10 minutes) is crucial for reducing background.

• Detection: Both chemiluminescence and fluorescent detection systems are compatible with CLDND1 antibodies.

• Expected results: CLDND1 should appear as a band at approximately 29 kDa .

How can CLDND1 antibodies be used to investigate tight junction integrity in disease models?

CLDND1 antibodies provide valuable tools for investigating tight junction (TJ) integrity in disease models:

• Co-localization studies: Combine CLDND1 antibodies with antibodies against other TJ proteins (occludin, ZO-1) using multi-color immunofluorescence to assess TJ complex integrity. Disruption of co-localization often indicates compromised barrier function.

• Disease progression monitoring: Track changes in CLDND1 expression and localization during disease progression. For instance, in colorectal cancer, CLDND1 shifts from predominantly cytoplasmic to membrane localization .

• Therapeutic intervention assessment: Use CLDND1 immunostaining to evaluate the effectiveness of interventions designed to restore barrier function.

• Live cell imaging: Conjugate CLDND1 antibodies to non-disruptive fluorophores for live cell imaging of tight junction dynamics in responsive cell models.

• Correlative analysis: Correlate CLDND1 expression/localization with functional barrier measurements (TEER, paracellular flux) to establish structure-function relationships.

In fibrotic disease models, researchers can use CLDND1 antibodies to specifically target nonjunctional CLDN1, which has been implicated in fibrogenesis across multiple organs including liver, lung, and kidney .

What is the significance of CLDND1 expression in cancer research and how can antibodies help characterize its role?

CLDND1 expression has emerged as a significant factor in cancer research, particularly in colorectal cancer (CRC):

• Differential expression analysis: CLDND1 is overexpressed in CRC compared to normal mucosa (p<0.001), with distinct subcellular localization patterns .

• Molecular subtyping: CLDND1 shows variable expression across CRC molecular subtypes, with highest expression in consensus molecular subtype CMS2, transit-amplifying, and C5 subtypes (all p<0.001) .

• Prognostic significance: Lower CLDND1 expression predicts better outcomes in C3 and C5 molecular subtypes of CRC (p=0.012 and p=0.004, respectively) .

• Therapeutic targeting: Monoclonal antibodies targeting CLDND1, such as the 6F6 mAb against the extracellular domain, have shown promising anti-tumor effects in preclinical models .

Researchers can use CLDND1 antibodies for:

• Tissue microarray analysis to correlate expression with clinical outcomes

• Functional studies in cancer cell lines to assess effects on proliferation, migration, and invasion

• In vivo imaging of CLDND1-positive tumors in animal models

• Screening potential therapeutic compounds that modulate CLDND1 expression or function

How can researchers utilize CLDND1 antibodies in therapeutic development?

Recent research suggests CLDND1 as a promising therapeutic target, particularly for fibrosis and cancer:

• Target validation: CLDND1 antibodies can help validate this protein as a therapeutic target through techniques like immunoprecipitation, ChIP, and immunohistochemistry to establish expression patterns in disease states.

• Antibody-based therapeutics: Highly specific monoclonal antibodies targeting conformation-dependent epitopes of exposed nonjunctional CLDN1 have shown therapeutic potential in liver fibrosis models . The 6F6 mAb against CLDN1's extracellular domain reduced survival, growth, and migration of CLDN1-positive CRC cells and decreased tumor growth and liver metastasis formation in mouse models .

• Combination therapy assessment: CLDND1 antibodies can help monitor changes in tight junction composition during combination therapies, potentially identifying synergistic approaches.

• Patient stratification biomarker: CLDND1 expression analysis using validated antibodies may help identify patients most likely to respond to tight junction-targeted therapies.

• Safety profiling: Studies with humanized anti-CLDN1 antibodies in nonhuman primates did not reveal serious adverse events even at high steady-state concentrations, supporting their further development .

How should researchers address potential cross-reactivity with other claudin family members?

The claudin family consists of at least 23 members with structural similarities, creating challenges for antibody specificity:

• Epitope selection: Choose antibodies raised against unique regions of CLDND1 with minimal sequence homology to other claudins.

• Comprehensive validation: Validate antibodies against cell lines overexpressing different claudin family members to confirm specificity.

• Multiple detection methods: Employ orthogonal techniques (Western blot, IHC, IF) to confirm findings, as cross-reactivity may manifest differently across methods.

• Genetic controls: Include CLDND1 knockout/knockdown samples as negative controls and CLDND1-overexpressing samples as positive controls.

• Pre-absorption controls: Pre-absorb antibodies with recombinant CLDND1 protein to demonstrate binding specificity.

• Batch testing: Test new antibody lots against reference standards to ensure consistent specificity.

• Species considerations: Note that cross-reactivity profiles may differ across species due to sequence variations in claudin family members.

What are the best practices for quantifying CLDND1 expression in experimental samples?

Accurate quantification of CLDND1 expression requires careful methodological considerations:

• Western blot quantification:

  • Include gradient standards of recombinant CLDND1 protein

  • Normalize to multiple housekeeping proteins

  • Use digital imaging systems with linear dynamic range

  • Report relative rather than absolute values

• Immunohistochemistry quantification:

  • Develop consistent scoring systems (H-score, Allred)

  • Use digital pathology software for unbiased assessment

  • Consider both staining intensity and percentage of positive cells

  • Distinguish between membrane, cytoplasmic, and nuclear staining

• qPCR for transcript analysis:

  • Design primers spanning exon-exon junctions

  • Validate primer efficiency and specificity

  • Use multiple reference genes for normalization

  • Consider potential splice variants

• Flow cytometry:

  • Optimize permeabilization for intracellular/membrane protein detection

  • Use median fluorescence intensity rather than mean

  • Include fluorescence-minus-one (FMO) controls

  • Consider fixation effects on epitope accessibility

How can researchers effectively investigate the relationship between CLDND1 and disease progression?

To establish meaningful connections between CLDND1 and disease progression:

• Longitudinal sampling:

  • Collect samples at multiple disease stages

  • Use paired samples when possible (pre/post-treatment)

  • Include adequate controls for each timepoint

• Multi-omics integration:

  • Correlate protein expression (antibody-based) with transcriptomic data

  • Integrate with clinical parameters and outcomes

  • Explore potential regulatory mechanisms affecting CLDND1

• Functional studies:

  • Use antibody-mediated neutralization to assess functional consequences

  • Develop cellular models with tunable CLDND1 expression

  • Employ 3D culture systems to better recapitulate in vivo conditions

• Animal models:

  • Select models that appropriately recapitulate human CLDND1 biology

  • Conduct intervention studies at different disease stages

  • Use tissue-specific knockout/knockin approaches when feasible

• Clinical correlation:

  • Stratify patients based on CLDND1 expression patterns

  • Correlate with treatment response and survival outcomes

  • Consider CLDND1 in the context of molecular subtypes, as shown in CRC research

How should researchers address inconsistent results between different CLDND1 detection methods?

When facing discrepancies between different detection methods:

• Method-specific considerations:

  • Western blot detects denatured protein, potentially missing conformation-dependent epitopes

  • IHC preserves tissue architecture but may suffer from epitope masking

  • IF offers superior resolution but may lack sensitivity for low-abundance targets

  • ELISA provides quantitative data but lacks spatial information

• Systematic validation approach:

  • Test multiple antibodies targeting different epitopes

  • Compare results across cell/tissue types with known CLDND1 expression

  • Include genetic controls (overexpression/knockdown)

  • Consider post-translational modifications affecting epitope recognition

• Biological explanations:

  • Splice variants may be detected differently by various antibodies

  • Subcellular localization changes (cytoplasmic vs. membrane) affect detection efficiency

  • Protein-protein interactions may mask epitopes in specific contexts

• Technical resolution strategies:

  • Optimize sample preparation for each method separately

  • Document all experimental conditions thoroughly

  • Consider orthogonal methods that don't rely on antibodies (MS, RNA-seq)

  • Report discrepancies transparently in publications

What are the key considerations when interpreting CLDND1 localization patterns in different tissue types?

Interpreting CLDND1 localization requires consideration of several factors:

• Normal versus pathological tissue:

  • In normal tissues, CLDND1 is predominantly cytoplasmic

  • In colorectal cancer, CLDND1 shows increased membrane localization

  • Changes in localization may precede changes in expression level

• Tissue-specific expression patterns:

  • Baseline expression varies significantly between tissue types

  • Cellular heterogeneity within tissues affects interpretation

  • Compare with known expression databases for validation

• Junctional versus non-junctional localization:

  • Distinguish between CLDND1 incorporated into tight junctions and non-junctional pools

  • Non-junctional CLDND1 (njCLDN1) appears to have distinct functions in processes like fibrosis

  • Co-staining with other junction proteins helps distinguish these populations

• Technical considerations:

  • Fixation methods affect membrane protein preservation

  • Antigen retrieval techniques influence epitope accessibility

  • Section thickness impacts visualization of membrane structures

  • Super-resolution microscopy may be necessary to distinguish subtle localization differences