CDH13 Antibody

What is CDH13 and what are its molecular characteristics?

CDH13 (cadherin 13, also known as H-cadherin or T-cadherin) is an atypical member of the cadherin family of transmembrane glycoproteins that mediate calcium-dependent cell-cell adhesion. Unlike typical cadherins, CDH13 has unique structural properties:

• Full Name: cadherin 13, H-cadherin (heart)

• Calculated Molecular Weight: 713 amino acids, 78 kDa

• Observed Molecular Weight: 105 kDa (note the difference from calculated weight, suggesting post-translational modifications)

• Gene ID (NCBI): 1012

• UniProt ID: P55290

CDH13 plays a critical role in maintaining normal tissue architecture and has been implicated in various biological processes including tumor neovascularization, apoptosis, and cell cycle regulation .

What applications are CDH13 antibodies typically used for?

CDH13 antibodies can be used in multiple experimental applications, with varying recommended dilutions:

It is strongly recommended to titrate each antibody in your specific experimental system to obtain optimal results, as performance can be sample-dependent .

What species reactivity is available for CDH13 antibodies?

Commercial CDH13 antibodies show varying reactivity profiles:

• Human and mouse reactivity is most common and well-validated

• Rat reactivity is available in select products

• Less common reactivities include canine, porcine, and monkey (based on gene sequence homology)

When selecting an antibody, it's important to verify that the specific reactivity has been experimentally confirmed rather than just predicted based on sequence homology. Cross-reactivity testing data should be reviewed, especially when working with less common model organisms .

How should CDH13 antibodies be stored and handled?

Optimal storage and handling conditions for CDH13 antibodies typically include:

• Storage temperature: -20°C (most products)

• Buffer composition: PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

• Stability: Generally stable for one year after shipment when properly stored

• Aliquoting: Often unnecessary for -20°C storage with glycerol-containing buffers

• Small volume formats (e.g., 20 μl) may contain 0.1% BSA as a stabilizer

Always check the manufacturer's specific recommendations as formulations can vary between suppliers.

How can researchers validate the specificity of CDH13 antibodies?

Validating antibody specificity is crucial for reliable research outcomes. For CDH13 antibodies, consider these validation approaches:

• Positive control tissues: Use mouse or human heart tissue, which consistently shows high CDH13 expression

• Cell line controls: L02 cells have been validated as positive controls

• Knockout/knockdown validation: Compare staining between CDH13 knockout or siRNA knockdown samples and wild-type samples

• Western blot band analysis: Verify the molecular weight (~105 kDa observed vs. 78 kDa calculated)

• Dual antibody validation: Use antibodies targeting different epitopes of CDH13 to confirm specificity

• Immunoprecipitation followed by mass spectrometry: To confirm the identity of the pulled-down protein

• Peptide competition assay: Pre-incubation with immunogen peptide should abolish specific signal

Careful documentation of validation steps strengthens research credibility and reproducibility.

What role does CDH13 play in cancer progression and how can antibodies help study this?

CDH13 has emerged as a significant factor in cancer biology, particularly in clear cell renal cell carcinoma (ccRCC):

• CDH13 is significantly upregulated in ccRCC compared to normal kidney tissues at both mRNA and protein levels

• Higher CDH13 expression correlates with better survival, lower cancer stages, and lower tumor grades in ccRCC patients

• CDH13 appears to play a crucial role in regulating the tumor microenvironment

• The epigenetic status of CDH13 is altered in cancer, with both DNA methylation and m6A modification affecting prognosis

CDH13 antibodies can be used to:

• Quantify expression levels in patient samples via IHC or Western blot

• Study association with clinicopathological features

• Investigate cell-cell interactions in tumor microenvironments

• Evaluate potential as a therapeutic target

When designing such studies, consider using multiple detection methods and correlating protein expression with genomic and transcriptomic data for a comprehensive understanding .

How does the tumor microenvironment affect CDH13 detection and what methodological approaches can overcome these challenges?

The tumor microenvironment presents several challenges for accurate CDH13 detection:

• Heterogeneous expression: CDH13 expression can vary significantly across different regions of a tumor, requiring multiple sampling

• Immune cell infiltration: CDH13 expression correlates with immune infiltration patterns, which can confound analysis

• Stromal contamination: Non-tumor cells may express different levels of CDH13

• Post-translational modifications: These can affect antibody binding and vary in different microenvironmental conditions

Methodological approaches to overcome these challenges:

• Single-cell analysis: Combining antibody-based detection with single-cell transcriptomics

• Multiplex immunofluorescence: Co-staining CDH13 with immune cell markers to distinguish cell-specific expression

• Laser capture microdissection: Isolating specific cellular populations before analysis

• Spatial transcriptomics: Correlating CDH13 protein expression with spatial gene expression data

• Quantitative image analysis: Using digital pathology tools to quantify expression levels rather than subjective scoring

Research has shown that comprehensive analysis using ssGSEA (single-sample Gene Set Enrichment Analysis) can quantify the infiltration level of immune cells and determine the correlation between CDH13 expression and immune infiltration .

What are the optimal methods for quantifying CDH13 expression in tissue samples?

Quantification of CDH13 expression in tissue samples requires standardized approaches for reliable results:

• Immunohistochemistry quantification:

  • Image-Pro Plus software (version 6.0) can be used to analyze immunohistochemistry images

  • Integral optical density (IOD)/Area measurements provide quantitative expression levels

  • Whole slide scanning with automated analysis reduces subjective bias

• Protein extraction optimization:

  • For Western blot analysis, optimized lysis buffers containing protease inhibitors are essential

  • Membrane fraction enrichment may improve detection of this membrane-associated protein

• RNA expression analysis:

  • qRT-PCR with validated primer sets

  • RNA-seq analysis with proper normalization

  • In situ hybridization for spatial context

• Reference standards:

  • Include gradient standards on each immunoblot

  • Use housekeeping proteins appropriate for the tissue type

  • Consider tissue microarrays for comparative analysis across multiple samples

• Validation across platforms:

  • Correlate protein levels from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database with immunohistochemistry findings

  • Compare with transcriptomic data from resources like The Cancer Genome Atlas (TCGA)

Research has demonstrated that these multi-modal approaches provide more reliable quantification than single-method analysis .

What are the optimal antigen retrieval methods for CDH13 immunohistochemistry?

Successful CDH13 immunohistochemistry depends heavily on appropriate antigen retrieval:

Primary recommendation:

• TE buffer pH 9.0 has been validated for mouse and human heart tissues

Alternative method:

• Citrate buffer pH 6.0 can also be effective

Protocol considerations:

• Temperature and time: Typically 95-100°C for 15-20 minutes, followed by natural cooling

• Tissue type matters: Different fixation times may require adjusted retrieval conditions

• Background reduction: Include appropriate blocking steps with serum matching the secondary antibody species

• Signal amplification: Consider tyramide signal amplification for low-abundance detection

For formalin-fixed, paraffin-embedded tissues, adequate deparaffinization and hydration prior to antigen retrieval are essential for consistent results. Following antigen retrieval, endogenous peroxidase blocking is necessary before applying the primary anti-CDH13 antibody (typically at 1:50-1:500 dilution) .

How can researchers troubleshoot weak or inconsistent Western blot signals for CDH13?

Western blotting for CDH13 can present challenges due to its molecular characteristics:

Common issues and solutions:

• Molecular weight discrepancy:

  • Expected calculated weight: 78 kDa

  • Observed weight: 105 kDa

  • This difference is due to post-translational modifications; ensure you're examining the correct band

• Protein extraction optimization:

  • Use RIPA or NP-40 based buffers with protease inhibitors

  • Consider membrane-enrichment protocols for improved yield

  • Extended lysis times may be necessary for complete extraction

• Transfer conditions:

  • Higher molecular weight proteins may require extended transfer times

  • Consider semi-dry vs. wet transfer optimization

  • PVDF membranes often yield better results than nitrocellulose for this protein

• Antibody dilution:

  • Start with manufacturer recommendations (typically 1:500-1:1000)

  • Perform titration experiments if signal is weak

  • Extended primary antibody incubation (overnight at 4°C) often improves results

• Positive controls:

  • Include mouse heart tissue, human heart tissue, or L02 cells as positive controls

  • Recombinant CDH13 protein can serve as an additional control

• Signal enhancement:

  • Enhanced chemiluminescence (ECL) substrates with varying sensitivity are available

  • Consider fluorescent secondary antibodies for more quantitative analysis

Adjust blocking conditions (5% BSA often works better than milk for some phosphorylated proteins) and secondary antibody concentrations as needed.

What considerations are important for dual immunofluorescence with CDH13 and other markers?

Dual immunofluorescence staining involving CDH13 requires careful planning:

• Antibody compatibility:

  • Primary antibodies must be from different host species (e.g., rabbit anti-CDH13 with mouse anti-marker)

  • If same-species antibodies are unavoidable, use directly conjugated antibodies or sequential immunostaining with thorough blocking

• Fluorophore selection:

  • Choose fluorophores with minimal spectral overlap

  • Consider tissue autofluorescence spectrum when selecting fluorophores

  • For CDH13 (often less abundant), use brighter fluorophores (e.g., Alexa Fluor 488 or 594)

• Protocol optimization:

  • Dilute CDH13 antibodies 1:50-1:500 for immunofluorescence

  • Perform antigen retrieval suitable for all target antigens

  • Include appropriate controls (single stains, isotype controls, blocking peptides)

• Confocal imaging considerations:

  • Adjust laser power and detector gain to avoid bleed-through

  • Collect images sequentially rather than simultaneously

  • Use spectral unmixing if available

• Quantitative analysis:

  • Use software that can quantify colocalization (e.g., Mander's coefficient, Pearson's correlation)

  • Establish thresholds based on control samples

For CDH13 in mouse heart tissue, validated protocols show good results with standard immunofluorescence approaches using rabbit polyclonal antibodies .

What are the key considerations for immunoprecipitation experiments targeting CDH13?

Successful immunoprecipitation (IP) of CDH13 requires attention to several factors:

• Antibody amount optimization:

  • Recommended range: 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

  • Titration experiments may be necessary for your specific sample type

• Lysis buffer selection:

  • Non-denaturing buffers containing 1% NP-40 or 0.5% Triton X-100 are typically effective

  • Include protease inhibitors and phosphatase inhibitors if studying phosphorylation

  • Buffer ionic strength affects antibody binding; optimize salt concentration

• Bead selection and pre-clearing:

  • Protein A/G beads work well with rabbit anti-CDH13 antibodies

  • Pre-clear lysates with beads alone to reduce non-specific binding

  • Consider magnetic beads for cleaner results and easier handling

• Controls:

  • Include isotype control antibody IP

  • Input sample (pre-IP lysate)

  • Mouse heart tissue has been validated as a positive control for CDH13 IP

• Elution and detection:

  • Gentle elution with sample buffer at 70°C may better preserve protein integrity than boiling

  • Western blot detection using a different anti-CDH13 antibody (targeting a different epitope) validates specificity

When analyzing CDH13 interaction partners, consider label-free mass spectrometry or targeted approaches to verify protein complexes.

How is CDH13 expression altered across different cancer types and what are the implications?

CDH13 expression patterns vary significantly across cancer types, with important clinical implications:

• Clear cell renal cell carcinoma (ccRCC):

  • Significantly upregulated compared to normal kidney tissues

  • Higher expression correlates with better survival, lower cancer stages, and lower tumor grades

  • May serve as a favorable prognostic marker

• Other cancer types (from broader literature):

  • Expression patterns can be context-dependent

  • Both upregulation and downregulation have been reported

  • Epigenetic silencing through methylation is common in some cancer types

  • May function as a tumor suppressor or oncogene depending on cancer type

• Clinical correlations:

  • The relationship between CDH13 expression and clinicopathological features (tumor stage, grade, etc.) varies by cancer type

  • CDH13 methylation status has prognostic significance

  • Both DNA methylation and m6A modification of CDH13 can affect patient outcomes

• Research applications:

  • Diagnostic biomarker potential

  • Prognostic indicator

  • Therapeutic target

  • Predictor of treatment response

Researchers should consider tissue-specific contexts when studying CDH13 in different cancer types, as its role appears to be highly dependent on the cellular environment and cancer type .

What is the relationship between CDH13 expression and immune infiltration in tumors?

CDH13 plays a significant role in regulating the tumor microenvironment, particularly immune cell infiltration:

• Correlation with immune cells:

  • CDH13 expression levels correlate with specific immune cell infiltration patterns

  • Single-sample Gene Set Enrichment Analysis (ssGSEA) can quantify this relationship

  • The Tumor and Immune System Interaction Database (TISIDB) allows Spearman's correlation analysis between CDH13 expression and tumor lymphocyte infiltration

• Immune regulatory mechanisms:

  • CDH13 may influence immunostimulators and immunoinhibitors

  • Affects major histocompatibility complex presentation

  • May modulate cytokine/chemokine profiles in the tumor microenvironment

• Methodological approaches:

  • Wilcoxon rank sum test can evaluate differences in immune cells between CDH13-high and CDH13-low cohorts

  • Multiplex immunohistochemistry can visualize spatial relationships

  • Flow cytometry can quantify immune subpopulations in relation to CDH13 expression

• Therapeutic implications:

  • CDH13-mediated immune regulation may influence immunotherapy response

  • Combined targeting strategies may be more effective in certain contexts

  • Immune infiltration patterns could serve as companion biomarkers for CDH13-targeted therapies

Understanding these complex relationships requires integrated multi-omic approaches, correlating protein expression with transcriptomic and spatial data .

How can researchers evaluate CDH13 as a potential therapeutic target in cancer?

Evaluating CDH13 as a therapeutic target requires systematic investigation across multiple dimensions:

• Target validation approaches:

  • Genetic manipulation (knockout/knockdown) to assess phenotypic effects

  • Correlation of expression with clinical outcomes across large patient cohorts

  • Assessment of downstream signaling pathways affected by CDH13 modulation

  • Evaluation of synergistic effects with standard treatments

• Therapeutic strategies to consider:

  • Monoclonal antibodies targeting CDH13

  • Small molecule inhibitors of CDH13-mediated signaling

  • Epigenetic modifiers to regulate CDH13 expression

  • CDH13-based immunotherapeutic approaches (CAR-T, bispecific antibodies)

• Biomarker development:

  • Companion diagnostics to identify patients likely to respond

  • Pharmacodynamic markers to confirm target engagement

  • Resistance biomarkers to predict treatment failure

• Preclinical models:

  • Patient-derived xenografts with varying CDH13 expression levels

  • Genetically engineered mouse models

  • 3D organoid cultures to better recapitulate in vivo conditions

• Translational considerations:

  • Investigation of potential off-target effects (CDH13 is expressed in heart tissue)

  • Delivery strategies to tumor sites

  • Combination approaches with standard of care treatments

The research evidence suggesting CDH13 as a novel prognostic biomarker and therapeutic target, particularly in ccRCC, provides a foundation for these investigations, though further validation across multiple cancer types is needed .

What methods are recommended for studying CDH13 methylation status in cancer samples?

CDH13 methylation status has significant implications for cancer biology and patient outcomes. Researchers should consider these methodological approaches:

• DNA methylation analysis techniques:

  • Bisulfite sequencing for comprehensive CpG site coverage

  • Methylation-specific PCR for targeted analysis of specific regions

  • Methylation arrays (e.g., Illumina MethylationEPIC) for genome-wide profiling

  • Reduced representation bisulfite sequencing (RRBS) for cost-effective genome-wide screening

• RNA modification analysis:

  • m6A methylation analysis using m6A-seq or miCLIP

  • Integration with transcriptomic data to assess functional consequences

  • RNA immunoprecipitation to study m6A writers/readers/erasers that interact with CDH13 mRNA

• Correlation with expression:

  • Integrated analysis of methylation status and protein/mRNA expression

  • Functional validation with demethylating agents (e.g., 5-azacytidine)

  • Reporter assays to confirm regulatory regions

• Clinical correlations:

  • Relation of methylation patterns to patient survival and disease progression

  • Association with response to specific therapies

  • Comparison across different cancer subtypes

• Bioinformatic resources:

  • The Cancer Genome Atlas (TCGA) methylation datasets

  • Gene Expression Omnibus (GEO) for methylation array data

  • cBioPortal for integrated genomic analyses

Research has shown that the prognosis of ccRCC patients is related not only to DNA methylation but also to m6A modification of CDH13, highlighting the importance of comprehensive epigenetic profiling .

What are the most important considerations when selecting a CDH13 antibody for specific research applications?

When selecting a CDH13 antibody for your research, consider these critical factors:

• Application compatibility:

  • Verify validation data for your specific application (WB, IHC, IF, IP, etc.)

  • Check recommended dilutions and protocols for your application

  • Review published literature using the antibody for similar applications

• Species reactivity:

  • Confirm experimental validation in your species of interest

  • Multiple species reactivity (human, mouse, rat) is available across different products

  • Consider epitope conservation if working with uncommon species

• Clonality and host:

  • Polyclonal antibodies (typically rabbit-derived) offer high sensitivity but may have batch variation

  • Monoclonal antibodies provide consistency across experiments

  • Host species matters for co-staining experiments

• Technical specifications:

  • Epitope location (C-terminal vs. other regions)

  • Format (unconjugated vs. conjugated)

  • Validation data quality and comprehensiveness

• Experimental validation:

  • Positive controls (mouse heart tissue, human heart tissue, L02 cells)

  • Expected molecular weight (observed: 105 kDa; calculated: 78 kDa)

  • Specific application protocols and troubleshooting guidelines