CDH4 Antibody

What is CDH4/R-cadherin and why is it significant in research?

CDH4 (Cadherin-4) is a 916-amino acid residue protein encoded by the CDH4 gene in humans. Also known as R-cadherin, it belongs to the cadherin family of calcium-dependent cell adhesion molecules. CDH4 plays crucial roles in axon guidance and cell adhesion processes, particularly during neural development. It is primarily localized to the cell membrane and contains glycosylated post-translational modifications. The protein is notably expressed in the brain but can also be found in other tissues. Its significance in research stems from its involvement in tissue development, cancer progression, and neurological functions .

What are the common synonyms and identifiers for CDH4 antibody targets?

When searching literature and databases for CDH4 antibodies, researchers should be aware of several synonyms and alternative identifiers:

• CDH4 (official gene symbol)

• Cadherin-4 (full protein name)

• R-cadherin or R-CAD (common alternative names)

• CAD4 (abbreviated form)

• RCAD (alternative abbreviation)

• Retinal cadherin (functional description)

The human CDH4 protein has a molecular weight of approximately 100 kDa (precisely 100,281 Da), which is important when validating antibody specificity in applications like Western blotting .

What are the primary applications for CDH4 antibodies in research?

CDH4 antibodies are utilized in multiple experimental techniques, including:

The antibody selection should be based on the specific application and sample type being analyzed .

How should CDH4 antibodies be stored and handled to maintain optimal activity?

For optimal performance and longevity of CDH4 antibodies, proper storage and handling are essential:

• Long-term storage: Store at -20°C for up to one year

• Short-term storage and frequent use: Store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles, which can cause protein denaturation and decreased antibody activity

• When using lyophilized antibodies, reconstitute according to manufacturer's instructions in sterile water or buffer

• Working solutions should be prepared fresh and kept on ice during experiments

• If antibodies are conjugated (with fluorophores, biotin, etc.), protect from light to prevent photobleaching

• Document lot numbers and purchase dates to track antibody performance over time

What is the recommended protocol for Western blot analysis using CDH4 antibodies?

The following protocol has been optimized for detecting CDH4 in Western blot applications:

• Sample preparation:

  • Prepare cell/tissue lysates (50 μg of protein per lane recommended)

  • Use reducing conditions for optimal detection

• Electrophoresis conditions:

  • Run on 5-20% SDS-PAGE gel

  • 70V for stacking gel, 90V for resolving gel

  • 2-3 hours running time

• Transfer conditions:

  • Transfer to nitrocellulose membrane

  • 150mA for 50-90 minutes

• Blocking:

  • Block with 5% non-fat milk in TBS

  • 1.5 hour at room temperature

• Primary antibody incubation:

  • Dilute CDH4 antibody to 0.5 μg/mL in blocking buffer

  • Incubate overnight at 4°C

• Washing:

  • Wash 3 times with TBS-0.1% Tween

  • 5 minutes per wash

• Secondary antibody incubation:

  • Anti-rabbit IgG-HRP at 1:5000 dilution

  • 1.5 hour at room temperature

• Detection:

  • Develop using enhanced chemiluminescence (ECL)

  • Expected band size for R-cadherin/CDH4: approximately 125 kDa

What controls should be included when validating CDH4 antibody specificity?

When validating CDH4 antibodies for research, multiple controls should be incorporated:

• Positive control samples: Use tissues/cells known to express CDH4 (brain tissue, U20S, A549, or U87 cell lines)

• Negative control samples: Include tissues/cells with minimal CDH4 expression

• Peptide competition assay: Pre-incubate antibody with the immunizing peptide before application to verify specific binding

• Isotype control: Use matched isotype antibody (e.g., rabbit IgG) to assess non-specific binding

• Knockout/knockdown validation: If possible, test antibody on CDH4 knockout or knockdown samples

• Multiple antibody validation: Use different antibodies targeting different epitopes of CDH4

• Secondary antibody-only control: Omit primary antibody to detect non-specific secondary antibody binding

These controls help confirm antibody specificity and reduce the likelihood of misinterpreting results due to cross-reactivity or non-specific binding .

What is the tissue distribution pattern of CDH4 expression?

CDH4 exhibits a distinct tissue expression pattern important for experimental design and interpretation:

This expression pattern should be considered when selecting positive control tissues and cell lines for CDH4 antibody validation and research applications .

How does CDH4 expression change in renal cell carcinoma (RCC) and what is its prognostic significance?

Research on CDH4 expression in renal cell carcinoma has revealed complex patterns with potential diagnostic and prognostic value:

These findings suggest CDH4 may have tumor suppressor functions in certain RCC contexts, with expression decreasing during disease progression and metastasis .

What methodologies are recommended for analyzing CDH4 expression in tissue samples?

Multiple complementary methods are recommended for comprehensive CDH4 expression analysis:

• mRNA expression analysis:

  • Real-time PCR for relative quantification

  • RNA-seq for genome-wide expression context

  • In situ hybridization for spatial distribution in tissues

• Protein expression analysis:

  • Immunohistochemistry with appropriate controls:

    • Heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

    • Blocking with 10% goat serum

    • Primary antibody concentration: 1μg/ml

    • Use of biotinylated secondary antibody with Strepavidin-Biotin-Complex

    • DAB as chromogen for visualization

  • Immunofluorescence for co-localization studies

  • Western blotting for semi-quantitative protein level assessment

  • Flow cytometry for cell-by-cell expression analysis

• Bioinformatic analysis:

  • TCGA database mining for expression correlation with clinical parameters

  • Survival analysis based on expression levels

  • Multi-omics correlation studies

This multi-method approach allows for cross-validation and comprehensive characterization of CDH4 expression patterns in normal and pathological samples .

What are common issues when working with CDH4 antibodies and how can they be resolved?

For challenging samples, consider specialized techniques like signal amplification methods or more sensitive detection systems .

How can researchers determine the optimal antibody concentration for different applications?

Determining optimal antibody concentration requires systematic titration:

• Western blot titration:

  • Begin with 3-5 concentrations (e.g., 0.1, 0.5, 1.0, 2.0, 5.0 μg/ml)

  • Use consistent positive control sample across titrations

  • Select concentration that provides best signal-to-noise ratio

  • For CDH4, 0.5 μg/ml has been found effective for many cell lysates

• Immunohistochemistry/Immunofluorescence titration:

  • Test 4-5 dilutions on positive control tissues

  • Begin with manufacturer's recommended range (typically 1-10 μg/ml)

  • Evaluate both signal intensity and background

  • For CDH4 IHC, 1 μg/ml has shown good results in human glioma tissue

• Flow cytometry titration:

  • Test antibody at 0.25-5 μg per 10^6 cells

  • Compare signal separation between positive and negative populations

  • For CDH4, 1 μg per 10^6 cells has been effective for U20S cells

Document optimal conditions for each application and sample type in laboratory protocols to ensure consistency across experiments .

What criteria should be used to evaluate antibody performance in research applications?

Comprehensive antibody validation requires assessment across multiple parameters:

• Specificity evaluation:

  • Single band of expected size in Western blot

  • Absence of signal with peptide competition

  • Reduced/absent signal in knockdown/knockout samples

  • Consistent staining pattern with multiple antibodies to the same target

• Sensitivity assessment:

  • Detection limit determination

  • Ability to detect endogenous levels in relevant samples

  • Signal-to-noise ratio quantification

• Reproducibility testing:

  • Intra- and inter-assay consistency

  • Lot-to-lot variation analysis

  • Results consistency across different users

• Application-specific performance:

  • For WB: band clarity, minimal background, correct molecular weight

  • For IHC/IF: expected cellular localization, minimal background, correlation with literature

  • For Flow: clear population separation, minimal autofluorescence interference

• Consistency with orthogonal methods:

  • Correlation between protein detection and mRNA levels

  • Agreement with previously published expression patterns

Thorough documentation of these validation parameters enhances research reliability and reproducibility .

How can CDH4 antibodies be used to investigate the role of R-cadherin in cancer progression?

CDH4 antibodies enable multifaceted investigation of R-cadherin's role in cancer:

• Expression profiling across cancer stages:

  • IHC analysis of tumor tissue microarrays to correlate expression with clinical parameters

  • Flow cytometry to quantify expression in patient-derived samples

  • Western blot analysis of paired normal-tumor samples

• Functional studies:

  • Immunoprecipitation to identify CDH4 binding partners in cancer cells

  • Chromatin immunoprecipitation (ChIP) to study epigenetic regulation of CDH4

  • Blocking antibodies to disrupt CDH4 function in cell culture models

• Mechanistic investigations:

  • Co-localization studies with other adhesion molecules and signaling proteins

  • Analysis of epithelial-mesenchymal transition (EMT) markers in relation to CDH4 expression

  • Investigation of CDH4's role in cancer cell migration and invasion

• Translational applications:

  • Development of CDH4-based biomarkers for early detection

  • Correlation of CDH4 expression with treatment response

  • Assessment of CDH4 as a potential therapeutic target

These approaches can be particularly valuable in renal cell carcinoma research, where CDH4 expression gradually decreases with disease progression and correlates with patient survival .

What experimental approaches can be used to study CDH4's role in axon guidance and neural development?

Investigating CDH4's neurological functions requires specialized techniques:

• Developmental expression analysis:

  • Immunohistochemistry on brain tissue sections at different developmental stages

  • Western blot analysis of brain region-specific lysates across developmental timepoints

  • In situ hybridization combined with IHC for mRNA-protein correlation

• Axon guidance studies:

  • Growth cone collapse assays using CDH4 antibodies

  • Stripe assays with immobilized CDH4 proteins/antibodies

  • Time-lapse imaging of neuronal cultures with fluorescently labeled CDH4 antibodies

• Cell-cell interaction analysis:

  • Co-culture systems with CDH4-expressing and non-expressing cells

  • Adhesion assays using CDH4 antibodies as blocking agents

  • Atomic force microscopy to measure CDH4-mediated adhesion forces

• In vivo functional studies:

  • Intracerebroventricular injection of CDH4 antibodies

  • Ex vivo brain slice cultures with antibody treatment

  • Correlation of CDH4 expression with neural circuit formation

• Molecular interaction studies:

  • Co-immunoprecipitation to identify neuronal CDH4 binding partners

  • Proximity ligation assays to detect in situ protein interactions

  • FRET analysis of CDH4 interactions with other guidance molecules

These approaches can provide insights into how CDH4 contributes to the precise wiring of neural circuits during development .

What methods can be used to study post-translational modifications of CDH4 using specific antibodies?

Investigating CDH4 post-translational modifications requires specialized antibodies and techniques:

• Glycosylation analysis:

  • Western blotting before and after glycosidase treatment

  • Lectin affinity purification followed by CDH4 immunoblotting

  • Mass spectrometry of immunoprecipitated CDH4 to identify glycosylation sites

• Phosphorylation studies:

  • Phospho-specific CDH4 antibodies for Western blotting and IHC

  • Phosphatase treatment controls to verify phospho-specific signals

  • Kinase inhibitor treatments to identify regulatory pathways

• Proteolytic processing analysis:

  • Antibodies targeting different epitopes to detect full-length vs. cleaved forms

  • Cell-free proteolysis assays with recombinant CDH4 and candidate proteases

  • Protease inhibitor treatments to identify endogenous processing mechanisms

• Ubiquitination and degradation studies:

  • Co-immunoprecipitation with ubiquitin antibodies

  • Proteasome inhibitor treatments to assess CDH4 stability

  • Cycloheximide chase experiments with CDH4 immunoblotting

• Subcellular trafficking analysis:

  • Biotinylation of cell surface proteins followed by CDH4 immunoprecipitation

  • Immunofluorescence with organelle markers to track CDH4 localization

  • Live-cell imaging with fluorescently tagged CDH4 antibody fragments

Understanding these modifications is crucial as they regulate CDH4's adhesive properties, stability, and signaling functions in both normal development and disease states .