CDH1 Antibody, HRP conjugated

What is CDH1 protein and why is it a significant target for antibody-based detection in research?

CDH1 (E-cadherin) is a classical cadherin from the cadherin superfamily. It functions as a calcium-dependent cell-cell adhesion glycoprotein comprised of five extracellular cadherin repeats, a transmembrane region, and a highly conserved cytoplasmic tail. The protein plays critical roles in:

• Maintaining epithelial tissue integrity and barrier function

• Regulating cell-cell adhesions, mobility, and proliferation of epithelial cells

• Cancer progression when its function is lost or diminished

CDH1 mutations are correlated with gastric, breast, colorectal, thyroid, and ovarian cancers. Loss of function is thought to contribute to cancer progression by increasing proliferation, invasion, and/or metastasis . Because of its importance in normal tissue architecture and its role in cancer development, CDH1 is an important target for antibody-based detection in various research applications.

What are the primary applications for CDH1 HRP-conjugated monoclonal antibodies?

CDH1 HRP-conjugated monoclonal antibodies are primarily used in the following applications:

• Western Blotting (WB): For detecting CDH1 protein expression levels in cell or tissue lysates (recommended dilution 1:2000)

• Immunohistochemistry (IHC): For visualizing CDH1 localization in tissue sections (recommended dilution 1:150)

These applications provide researchers with tools to:

• Determine protein expression levels in different experimental conditions

• Examine subcellular localization patterns

• Assess protein function in normal and pathological tissues

The specificity of monoclonal antibodies like clone OTI2F9 enables consistent and reproducible results across experiments.

How should researchers optimize antibody dilution when using CDH1 HRP-conjugated antibodies for immunohistochemistry?

Optimizing antibody dilution for CDH1 HRP-conjugated antibodies requires systematic testing:

• Start with manufacturer's recommendation: Begin with the suggested dilution (e.g., 1:150 for IHC)

• Perform dilution series: Test a range of dilutions (e.g., 1:50, 1:150, 1:300, 1:600)

• Use appropriate controls:

  • Positive control (tissue known to express CDH1, such as epithelial tissues)

  • Negative control (tissue without CDH1 expression)

  • Antibody control (primary antibody omitted)

• Evaluate staining patterns: The optimal dilution should show:

  • Strong signal at cell-cell junctions/membrane localization

  • Low background staining

  • Clear distinction between positive and negative cells

For heat-induced epitope retrieval, use 10 mM citrate buffer pH 6.0 as this has been validated for CDH1 detection in tissues . The final protocol should produce consistent membranous staining in epithelial tissues without significant background.

What is the recommended protocol for using CDH1 antibodies in co-immunoprecipitation experiments?

For co-immunoprecipitation with CDH1 antibodies, follow this validated protocol:

• Cell preparation:

  • Transfect cells with constructs of interest (if studying interactions)

  • Pretreat with proteasome inhibitors (e.g., 500 nM epoxomicin for 6h) to stabilize protein complexes

• Lysis procedure:

  • Lyse cells in RIPA buffer containing protease inhibitors

  • Centrifuge at 10,000 × g for 10 min to clear lysates

• Immunoprecipitation:

  • Divide supernatant and incubate with CDH1 antibody for 2h at 4°C

  • Add protein G-agarose beads and incubate for 1h

  • Pull down protein G beads and wash 4 times with RIPA buffer

  • Elute bound proteins by boiling in 2× Laemmli buffer

• Detection:

  • Perform Western blot analysis using HRP-conjugated secondary antibody (if using non-conjugated primary)

  • For direct detection, use HRP-conjugated CDH1 antibody at 1:2000 dilution

This protocol has been successfully used to demonstrate protein-protein interactions involving cadherins and can be adapted for studying CDH1 binding partners.

What are common causes of non-specific binding when using CDH1 HRP-conjugated antibodies and how can they be minimized?

Common causes of non-specific binding and their solutions:

When specifically working with HRP-conjugated antibodies, it's critical to protect the enzyme activity by:

• Avoiding repeated freeze-thaw cycles

• Using stabilizing reagents such as 50% glycerol in storage buffer

• Working efficiently to minimize time at room temperature

How can researchers distinguish between true CDH1 signals and artifacts in immunohistochemistry applications?

Distinguishing true CDH1 signals from artifacts requires careful analysis and appropriate controls:

• Localization pattern analysis:

  • True CDH1 signal: Predominantly membranous staining at cell-cell junctions in epithelial tissues

  • Artifacts: Diffuse cytoplasmic staining, nuclear staining, or staining in tissues known to be CDH1-negative

• Essential controls:

  • Serial dilution test to demonstrate signal reduction with dilution

  • Peptide competition assay where pre-incubation with immunogenic peptide should abolish specific staining

  • Comparative analysis with alternative CDH1 antibody clones

  • Correlation with mRNA expression data from the same tissue

• Sample preparation considerations:

  • Proper fixation (typically 10% neutral buffered formalin)

  • Optimal antigen retrieval (citrate buffer pH 6.0)

  • Counterstaining with Mayer's hemalum solution for cellular context

Researchers should be aware that loss of CDH1 expression can be a real biological phenomenon in cancer progression, so absence of staining in tumor tissue alongside positive staining in adjacent normal epithelium may represent true downregulation rather than an artifact.

How can CDH1 antibodies be utilized in studying epithelial-mesenchymal transition (EMT) in cancer progression?

CDH1 antibodies are powerful tools for studying EMT in cancer progression:

• Protein expression profiling:

  • Use HRP-conjugated CDH1 antibodies in Western blot to quantify expression levels during EMT

  • Create progressive time-course studies of CDH1 downregulation following EMT induction

  • Compare with upregulation of mesenchymal markers (N-cadherin, vimentin)

• Immunohistochemical analysis:

  • Examine CDH1 localization and expression patterns at invasion fronts in tumor samples

  • Identify regions of partial or complete EMT based on CDH1 expression patterns

  • Correlate CDH1 loss with patient outcomes and metastatic potential

• Mechanistic studies:

  • Combine with antibodies against transcriptional repressors (SNAI1, TWIST, ZEB1/2)

  • Co-stain for β-catenin to analyze alterations in adherens junction complexes

  • Examine post-translational modifications of CDH1 during EMT initiation

• Clinical correlations:

  • Correlate CDH1 loss detected by antibody staining with CDH1 mutations identified by sequencing

  • Develop scoring systems for CDH1 expression patterns in tumors (complete loss vs. cytoplasmic relocalization)

Loss of CDH1 function is thought to contribute to cancer progression by increasing proliferation, invasion, and/or metastasis, making it a critical marker for tracking EMT progression in research models .

What are the experimental considerations when designing studies to investigate CDH1 protein-protein interactions using HRP-conjugated antibodies?

When investigating CDH1 protein-protein interactions with HRP-conjugated antibodies, researchers should consider:

• Preservation of protein complexes:

  • Use mild lysis conditions (0.5% NP-40, 20 mM Tris, pH 7.4, 100 mM NaCl₂, 0.5 mM EDTA)

  • Include proteasome inhibitors to stabilize protein complexes

  • Maintain samples at 4°C throughout processing

• Interaction validation approaches:

  • Reciprocal co-immunoprecipitation experiments

  • GST pull-down assays with recombinant proteins

  • Proximity ligation assays for in situ detection

• Controls for specificity:

  • IgG control immunoprecipitations

  • Competition with excess immunogenic peptide

  • Inclusion of known CDH1 interaction partners as positive controls

• Detection strategies:

  • Direct detection using HRP-conjugated CDH1 antibody

  • Two-step detection with primary antibody followed by HRP-conjugated secondary

  • Consider using antibody pairs recognizing different epitopes to confirm interactions

• Functional validation:

  • Correlate binding with functional outcomes (e.g., ubiquitination status)

  • Design domain deletion constructs to map interaction interfaces

  • Use site-directed mutagenesis to disrupt specific binding motifs

The cytoplasmic domain of CDH1 interacts with multiple proteins, including catenins that link to the actin cytoskeleton. These interactions are crucial for CDH1's role in maintaining epithelial integrity .

How can CDH1 HRP-conjugated antibodies be integrated into multiplexed assays for comprehensive analysis of cell adhesion mechanisms?

Integration of CDH1 HRP-conjugated antibodies into multiplexed assays requires strategic planning:

• Sequential immunohistochemistry approaches:

  • Use HRP-conjugated CDH1 antibody as the first detection reagent

  • Develop with a distinct chromogen (e.g., DAB for brown color)

  • Perform heat-mediated stripping of antibodies

  • Apply subsequent antibodies with different detection systems (e.g., alkaline phosphatase)

• Multiplex Western blotting strategies:

  • Strip and reprobe membranes after CDH1 detection

  • Use size-separated regions of the same blot for different targets

  • Employ spectral unmixing for distinguishing multiple fluorescent signals

• Protein array applications:

  • Include CDH1 antibody in antibody arrays for adhesion molecule profiling

  • Apply to lysates from different experimental conditions or tissue types

  • Quantify relative expression alongside other adhesion molecules

• Co-analysis with signaling pathway components:

  • Combine with detection of Rho GTPases which are regulated by CDH1 complexes

  • Examine APC/Cdh1 pathway components and their relationship to adhesion dynamics

  • Analyze β-catenin localization as an indicator of adhesion versus signaling functions

• Data integration approaches:

  • Correlate protein expression data with gene expression analysis

  • Create computational models of adhesion complex stoichiometry

  • Develop quantitative image analysis workflows for spatial protein relationships

When designing multiplexed assays, researchers should carefully validate that the CDH1 HRP-conjugated antibody signal is not affected by the presence of other detection reagents in the system.

How can CDH1 antibodies contribute to studying the relationship between cell adhesion and the anaphase-promoting complex/cyclosome in cell cycle regulation?

Recent research has revealed intriguing connections between CDH1/E-cadherin (the adhesion molecule) and Cdh1 (the anaphase-promoting complex/cyclosome activator), which can be explored using specialized antibody approaches:

• Distinguishing between the two Cdh1 proteins:

  • Use epitope-specific CDH1/E-cadherin antibodies targeting the extracellular domain

  • Apply APC/Cdh1-specific antibodies that recognize unique regions of the APC activator

  • Perform careful co-localization studies to map their distinct subcellular distributions

• Investigating functional relationships:

  • Examine how cell-cell contact via E-cadherin influences APC/Cdh1 activity

  • Study whether APC/Cdh1-mediated degradation of targets affects adhesion dynamics

  • Analyze cell cycle progression in the context of cell-cell adhesion status

• Mechanistic studies:

  • Use CDH1 antibodies to immunoprecipitate complexes and analyze for APC components

  • Conduct time-course studies during cell cycle progression to track both proteins

  • Employ antibodies in super-resolution microscopy to visualize potential co-localization events

APC/Cdh1 regulates Rho activity via targeting p190 for degradation, potentially influencing cell cytoskeleton organization that might affect E-cadherin-based adhesions . This connection offers a fascinating area for research at the intersection of cell cycle control and adhesion dynamics.

What methodological approaches can be used to study post-translational modifications of CDH1 using specialized antibodies?

Studying post-translational modifications (PTMs) of CDH1 requires sophisticated antibody-based approaches:

• PTM-specific antibody development and application:

  • Phospho-specific antibodies targeting known CDH1 phosphorylation sites

  • Antibodies recognizing ubiquitinated CDH1 to study degradation mechanisms

  • Glycosylation-sensitive antibodies to examine maturation state

• Enrichment strategies:

  • Two-step immunoprecipitation: first with total CDH1 antibody, then with PTM-specific antibody

  • Phospho-peptide enrichment followed by CDH1 antibody detection

  • Lectin affinity purification coupled with CDH1 antibody detection for glycosylation studies

• Functional correlation approaches:

  • Compare PTM status with membrane localization using fractionation and antibody detection

  • Analyze relationships between phosphorylation status and binding to catenins

  • Study ubiquitination patterns in relation to internalization and recycling

• Temporal dynamics:

  • Pulse-chase experiments with PTM induction followed by time-course antibody detection

  • Cell cycle synchronization with phase-specific PTM analysis

  • Stimulus-response studies examining rapid PTM changes

The cytoplasmic domain of CDH1 is subject to various modifications that regulate its stability and function. For instance, the APC/Cdh1 complex has been shown to mediate ubiquitination events that can be detected using specialized antibody approaches .

How can researchers develop quantitative assays using CDH1 HRP-conjugated antibodies to measure the dynamics of epithelial barrier function?

Quantitative assays using CDH1 HRP-conjugated antibodies for epithelial barrier studies:

• In situ barrier integrity assessment:

  • Surface biotinylation coupled with CDH1 antibody staining to correlate adhesion with barrier function

  • Calcium-switch assays with time-course CDH1 antibody detection to measure junction reassembly

  • Correlation of transepithelial electrical resistance (TEER) measurements with CDH1 expression quantification

• High-throughput screening approaches:

  • Automated image analysis of CDH1 antibody staining patterns in response to compound libraries

  • ELISA-based quantification of surface-accessible versus total CDH1 using non-permeabilized/permeabilized conditions

  • Flow cytometry for surface CDH1 quantification in response to barrier-modulating treatments

• Advanced microscopy techniques:

  • FRAP (Fluorescence Recovery After Photobleaching) studies with fluorescently-labeled CDH1 antibody fragments

  • Single-molecule tracking of CDH1 using quantum dot-conjugated antibodies

  • Super-resolution microscopy to quantify nanoscale organization of CDH1 clusters

• Mathematical modeling integration:

  • Develop algorithms correlating CDH1 staining intensity/patterns with predicted barrier strength

  • Create computational models that integrate multiple adhesion markers detected by multiplexed antibody approaches

  • Design machine learning approaches to classify barrier status based on complex CDH1 distribution patterns

These approaches leverage the specificity of CDH1 antibodies to provide quantitative insights into the functional status of epithelial barriers, which is relevant for studies of development, disease, and therapeutic interventions targeting epithelial function.

What are the essential validation procedures to confirm CDH1 antibody specificity before conducting critical experiments?

Comprehensive validation of CDH1 antibody specificity requires multiple approaches:

• Genetic validation:

  • Testing on CDH1 knockout/knockdown cellular models

  • Comparison of signal in CDH1-positive vs. CDH1-negative cell lines

  • Correlation with overexpression systems using tagged CDH1 constructs

• Biochemical validation:

  • Western blot showing a single band at the expected molecular weight (94.8 kDa)

  • Peptide competition assays demonstrating signal elimination

  • Cross-validation with multiple antibody clones recognizing different epitopes

• Application-specific validation:

  • For immunohistochemistry: Staining pattern analysis (membrane localization at cell-cell junctions)

  • For flow cytometry: Comparison with isotype controls and unstained samples

  • For co-immunoprecipitation: Verification of enrichment relative to input

• Species reactivity assessment:

  • Testing on tissues from different species if cross-reactivity is claimed

  • Sequence alignment of epitope regions across species

  • Titration curves for each species to determine optimal concentrations

• Documentation requirements:

  • Detailed records of lot-to-lot validation

  • Images of control experiments with positive and negative samples

  • Quantitative metrics of specificity and sensitivity

Researchers should insist on validation data from suppliers and conduct their own validation with relevant experimental systems before using CDH1 antibodies in critical experiments .

How should researchers properly prepare and store CDH1 HRP-conjugated antibodies to maintain optimal activity?

Proper handling and storage of CDH1 HRP-conjugated antibodies is critical for maintaining enzymatic activity and antibody specificity:

• Storage conditions:

  • Store at -20°C as received in buffer containing 50% glycerol

  • Avoid repeated freeze-thaw cycles (aliquot upon first thaw)

  • Protect from light to prevent photobleaching of potential fluorescent components

• Working solution preparation:

  • Thaw aliquots on ice

  • Dilute in appropriate buffer just before use

  • For Western blotting: Dilute to 1:2000 in blocking buffer

  • For IHC: Dilute to 1:150 in antibody diluent

• Stability considerations:

  • HRP-conjugated antibodies are typically stable for 12 months from receipt when properly stored

  • Working solutions should be prepared fresh and not stored for extended periods

  • Include HRP activity controls when using antibodies approaching expiration date

• Protection of HRP activity:

  • Avoid azide in buffers used with HRP-conjugated antibodies

  • Protect from oxidizing and reducing agents

  • Maintain recommended pH range (typically pH 7.3-7.5)

• Shipping and temporary storage:

  • Transport on blue ice or with cold packs

  • Temporary storage (1-2 weeks) can be at 4°C if the antibody contains 50% glycerol

  • Return to -20°C for long-term storage