CDH1 Recombinant Monoclonal Antibody

What is CDH1 and why are recombinant monoclonal antibodies used to study it?

CDH1 (E-cadherin) is a classical cadherin from the cadherin superfamily that functions as a calcium-dependent cell-cell adhesion glycoprotein. The protein comprises five extracellular cadherin repeats, a transmembrane region, and a highly conserved cytoplasmic tail . It plays essential roles in maintaining epithelial tissue integrity and is involved in cell proliferation, differentiation, migration, and apoptosis .

Recombinant monoclonal antibodies offer several advantages over traditional hybridoma-derived antibodies when studying CDH1:

• Higher batch-to-batch reproducibility through recombinant expression

• Reduction of animal usage in research

• Improved specificity through engineered binding domains

• Greater consistency in experimental results across long-term studies

The recombinant approach involves inserting CDH1 antibody genes into plasmid vectors and introducing these into appropriate host cells for expression using exogenous protein expression technology .

What are the primary applications for CDH1 recombinant monoclonal antibodies in research?

CDH1 recombinant monoclonal antibodies can be utilized in multiple experimental techniques:

The optimal working dilution should be determined by each researcher based on their specific experimental conditions .

How should CDH1 recombinant monoclonal antibodies be stored and handled to maintain optimal activity?

Proper storage and handling are critical for maintaining antibody functionality:

• Store at -20°C or -80°C for long-term preservation

• Aliquot to avoid repeated freezing and thawing cycles, which can degrade antibody quality

• Most preparations contain preservatives such as sodium azide (0.02%) and stabilizers like glycerol (50%), which should be considered when designing experiments

• When working with these antibodies, note that some preparations contain sodium azide, which is hazardous and should be handled by trained personnel only

• For immunohistochemistry applications on formalin-fixed, paraffin-embedded sections, validate antigen retrieval methods specifically for CDH1 detection

Detailed storage information should be consulted for each specific antibody product to ensure optimal performance and longevity .

What controls should be included when using CDH1 recombinant monoclonal antibodies in experiments?

Rigorous experimental design requires appropriate controls:

• Positive control: Cell lines or tissues known to express CDH1 (e.g., epithelial cell lines, normal breast tissue)

• Negative control: Cell lines or tissues with minimal CDH1 expression (e.g., certain mesenchymal cell lines)

• Isotype control: Matched recombinant antibody of the same isotype (commonly IgG1κ for mouse-derived clones or IgG for rabbit-derived clones)

• Secondary antibody-only control: To assess non-specific binding

• Knockdown/knockout validation: When possible, use CDH1 knockdown or knockout samples to confirm specificity

• Cross-reactivity assessment: Test against closely related proteins in the cadherin family

These controls help ensure the specificity and validity of experimental results, particularly when characterizing novel systems or applying the antibody to new research contexts.

How can epitope-specific CDH1 recombinant monoclonal antibodies be used to investigate different functional domains of the protein?

CDH1 contains distinct functional domains with specialized roles in cell adhesion and signaling:

• Extracellular domain antibodies (targeting cadherin repeats) can be used to study homophilic interactions and calcium-dependent adhesion

• Transmembrane domain-proximal antibodies can assess membrane localization and stability

• Cytoplasmic domain-specific antibodies can investigate interactions with catenins and cytoskeletal components

When designing experiments to investigate specific CDH1 functions:

• Select antibodies that recognize defined epitopes within the domain of interest

• Compare results using multiple epitope-specific antibodies to build a comprehensive understanding of domain function

• Combine with mutagenesis studies to correlate epitope accessibility with functional outcomes

• Consider conformation-specific antibodies that may only recognize properly folded CDH1

For example, antibodies targeting the extracellular domain can be used in live-cell blocking experiments to disrupt cell-cell adhesion, while cytoplasmic domain antibodies are better suited for co-immunoprecipitation studies of CDH1-associated protein complexes .

What methodological considerations should be addressed when using CDH1 recombinant monoclonal antibodies to study epithelial-mesenchymal transition (EMT)?

EMT involves the downregulation of CDH1, making it a critical marker for this process. When designing EMT studies:

• Temporal considerations: Establish a time-course to capture dynamic changes in CDH1 expression during EMT progression

  • Use flow cytometry with CDH1 antibodies to track population heterogeneity during transition

  • Combine with other EMT markers (N-cadherin, vimentin) for comprehensive phenotyping

• Spatial analysis: Employ immunohistochemistry to assess changes in CDH1 localization

  • Membrane-to-cytoplasmic translocation often precedes complete downregulation

  • Use confocal microscopy with CDH1 antibodies to examine subcellular redistribution

• Sensitivity optimization:

  • For detecting low CDH1 levels during early EMT, use signal amplification techniques

  • Consider proximity ligation assays to detect altered CDH1-catenin interactions

• Quantitative assessment:

  • Apply digital image analysis to IHC sections for objective quantification

  • Use western blotting with standard curves for precise measurement of expression changes

• Validation approach:

  • Correlate protein changes (antibody-based) with mRNA expression

  • Assess CDH1 promoter methylation status in parallel with protein expression

This comprehensive approach enables more nuanced insights into the functional implications of CDH1 dynamics during EMT progression.

How can contradictory results with CDH1 recombinant monoclonal antibodies be reconciled in cancer research applications?

Researchers may encounter discrepancies when studying CDH1 in cancer contexts. Methodological approaches to resolve these include:

• Clone-specific effects: Different antibody clones may recognize distinct epitopes that are differentially affected by:

  • Post-translational modifications

  • Proteolytic processing (e.g., generation of E-Cad/CTF2 fragment)

  • Protein-protein interactions that mask epitopes

  • Conformational changes in different cellular contexts

• Technical reconciliation strategies:

  • Use multiple antibody clones targeting different epitopes (e.g., comparing clone 1E1 with clone 3F4 )

  • Employ complementary detection methods (IHC, WB, FC) to build a comprehensive picture

  • Correlate antibody findings with genetic analyses (mutations, methylation)

  • Consider quantitative mass spectrometry as an antibody-independent validation

• Cancer-specific considerations:

  • CDH1 mutations in gastric, breast, colorectal, thyroid, and ovarian cancers may affect epitope availability

  • Heterogeneous expression within tumors requires careful sampling and analysis

  • Assess whether discrepancies reflect biological phenomena (e.g., partial EMT states) rather than technical artifacts

A systematic investigation comparing results across multiple antibodies, methods, and experimental conditions can help resolve apparent contradictions and reveal underlying biological complexity.

What are the optimal methodological approaches for using CDH1 recombinant monoclonal antibodies in multiplex immunoassays?

Multiplexing techniques allow researchers to simultaneously examine CDH1 alongside other proteins of interest:

• Multiplex flow cytometry considerations:

  • Select CDH1 antibody conjugates with minimal spectral overlap with other markers

  • Validate compensation controls specific for each fluorophore

  • Consider brightness hierarchies (pair brighter fluorophores with lower-expressed proteins)

• Multiplex immunohistochemistry/immunofluorescence optimization:

  • For sequential staining protocols, determine optimal antibody stripping conditions

  • For simultaneous staining, ensure antibodies are raised in different host species

  • Validate that multiplexing does not alter CDH1 staining pattern compared to single-stain controls

• Spatial biology applications:

  • For advanced platforms (e.g., imaging mass cytometry, multiplexed ion beam imaging):

    • Validate metal-conjugated CDH1 antibodies against traditional immunofluorescence

    • Optimize antibody concentration to prevent signal spillover

    • Develop computational approaches to quantify spatial relationships between CDH1 and other markers

• Proximity-based multiplexing:

  • For proximity ligation assays, carefully select antibody pairs that can simultaneously access adjacent epitopes

  • Validate specific interactions using known CDH1 binding partners (e.g., β-catenin)

These methodological considerations enable robust multiplexed analyses while maintaining the specificity and sensitivity of CDH1 detection.

How can CDH1 recombinant monoclonal antibodies be effectively used to study the role of E-cadherin in mechanotransduction?

E-cadherin (CDH1) serves as a mechanosensitive adhesion molecule that translates mechanical forces into biochemical signals. Advanced methodological approaches include:

• Force measurement techniques:

  • Combine CDH1 antibody visualization with atomic force microscopy

  • Use antibody-coated beads in optical tweezers experiments to apply defined forces to CDH1

  • Employ FRET-based tension sensors alongside antibody staining to correlate force with localization

• Cytoskeletal interactions:

  • Use CDH1 antibodies in co-localization studies with actin markers

  • Investigate how CDH1 promotes organization of radial actin fiber structure through interaction with AMOTL2

  • Apply CDH1 antibodies in proximity ligation assays to quantify interactions with cytoskeletal adaptor proteins

• Mechanical stimulation protocols:

  • Analyze CDH1 redistribution following cyclic stretch or shear stress

  • Assess changes in antibody epitope accessibility under different mechanical conditions

  • Quantify CDH1 clustering dynamics using super-resolution microscopy with antibody labeling

• Junction analysis:

  • Examine how CDH1 facilitates anchoring of radial actin fibers to junction complexes

  • Study the response of CDH1-based adhesions to contractile forces

  • Analyze recruitment of mechanosensitive proteins to CDH1 complexes under various mechanical conditions

These methodological approaches allow researchers to dissect the critical role of E-cadherin in cellular mechanobiology and tissue homeostasis.

What are the key considerations for using CDH1 recombinant monoclonal antibodies in studying blepharocheilodontic (BCD) syndrome and other CDH1-pathway related disorders?

BCD syndrome represents a rare autosomal dominant condition characterized by eyelid malformations, cleft lip/palate, and ectodermal dysplasia, which has been linked to the CDH1 pathway . When investigating such disorders:

• Genetic-protein correlation studies:

  • Use CDH1 antibodies to assess how specific mutations affect protein expression, localization, and function

  • Compare wild-type and mutant protein behavior in patient-derived samples or model systems

  • Employ antibodies in functional assays to evaluate adhesive properties of mutant CDH1

• Developmental context considerations:

  • Apply CDH1 antibodies to tissue sections from different developmental stages

  • Optimize immunohistochemistry protocols for embryonic and fetal tissues

  • Use multiple epitope-specific antibodies to ensure comprehensive detection of potentially truncated proteins

• Pathway analysis approaches:

  • Investigate both CDH1 and CTNND1 (p120-catenin) expression in parallel

  • Employ antibodies in co-immunoprecipitation studies to assess how mutations affect protein-protein interactions

  • Develop quantitative assays to measure the impact of mutations on CDH1-dependent signaling

• Translational research methodology:

  • Standardize antibody-based protocols for potential diagnostic applications

  • Develop tissue-specific staining algorithms for affected structures

  • Correlate antibody staining patterns with clinical severity metrics

These methodological considerations enable more precise characterization of CDH1 pathway disorders and may contribute to improved diagnostic and therapeutic approaches.