PCDH10 Antibody

What is PCDH10 and why is it significant in research?

PCDH10 is a member of the non-clustered protocadherin family encoded by the PCDH10 gene located on human chromosome 4q28.3. The canonical human PCDH10 protein consists of 1040 amino acid residues with a molecular mass of 112.9 kDa and is primarily localized in the cell membrane. This protein plays crucial roles in cell-cell adhesion and nervous system development .

The significance of PCDH10 in research stems from its dual importance in neurological development and tumor suppression. Initially studied in relation to neurological disorders such as autism, recent research has revealed its function as a tumor-suppressor gene in multiple cancer types, including hepatocellular carcinoma (HCC) . The protein undergoes alternative splicing, yielding two different isoforms, and exhibits tissue-specific expression patterns with high levels in all brain regions, testis, and ovary, while maintaining lower expression in other tissues .

What detection methods are most effective for PCDH10?

For reliable PCDH10 detection, Western blot (WB) and immunofluorescence (IF) techniques have proven most effective, with immunohistochemistry-paraffin (IHC-p) serving as a valuable complementary method . When conducting Western blot analysis, optimal results are achieved by:

• Extracting total protein 48-72 hours post-transfection or treatment

• Separating proteins using 8-10% polyacrylamide gel electrophoresis

• Transferring to PVDF membranes

• Blocking with 5% skim milk

• Incubating with primary PCDH10 antibodies overnight at 4°C

• Using HRP-conjugated secondary antibodies for visualization

For immunofluorescence applications, antibodies targeting different epitopes (particularly the C-terminal region) may provide better specificity for detecting distinct PCDH10 isoforms in neuronal or cancer cell lines .

How should researchers select the appropriate PCDH10 antibody for their experiments?

When selecting a PCDH10 antibody, researchers should consider:

For studies focusing on PCDH10's role in tumor suppression, antibodies specifically targeting the C-terminal region have demonstrated higher efficacy in detecting functional protein interactions with signaling pathway components like PI3K/Akt . Researchers investigating neuronal functions may benefit from antibodies recognizing extracellular domains involved in cell-cell adhesion .

What are the common challenges in using PCDH10 antibodies?

Common challenges researchers face when working with PCDH10 antibodies include:

• Specificity issues: Due to sequence homology with other protocadherin family members, cross-reactivity can occur, particularly with protocadherin alpha family members. Validation through knockout/knockdown controls is essential .

• Detection sensitivity: The relatively low endogenous expression of PCDH10 in some tissues necessitates optimization of antibody concentration and detection methods. Enhanced chemiluminescence systems are recommended for Western blot applications .

• Isoform discrimination: The presence of multiple PCDH10 isoforms from alternative splicing requires careful antibody selection to ensure detection of the specific isoform relevant to the research question .

• Post-translational modifications: Glycosylation of PCDH10 can affect antibody recognition. Treatment with deglycosylation enzymes prior to analysis may be necessary in some experimental contexts .

How can researchers design experiments to investigate PCDH10's role in tumor suppression pathways?

To investigate PCDH10's tumor suppression mechanisms, researchers should implement a multi-level experimental design:

• Expression modulation studies:

  • Overexpression: Transfect cells with pcDNA3.1-PCDH10 plasmid using Lipofectamine 2000 or similar transfection reagents

  • Knockdown: Utilize siRNA or shRNA targeting PCDH10

  • Confirm expression changes via RT-qPCR and Western blot analysis

• Functional assays:

  • Cell proliferation: Use CCK-8 assay at 24, 48, and 72 hours post-transfection

  • Colony formation: Plate cells at low density to assess long-term proliferative capacity

  • Cell cycle analysis: Employ flow cytometry to evaluate G1/G2 phase distribution

  • Apoptosis assessment: Use Annexin V/PI staining and flow cytometry

• Signaling pathway analysis:

  • Western blot for downstream effectors (p-AKT, p-MDM2, p53, p-GSK-3β, p21, caspase-3, Bax, Bcl-2, cyclin D1)

  • Co-immunoprecipitation (Co-IP) to identify protein-protein interactions, particularly with PI3K p85 subunit

  • Pathway inhibitor studies to confirm mechanistic relationships

• Validation in multiple cell lines: Test at least 2-3 different cell types relevant to the cancer being studied to ensure consistency of results .

What methodological approaches are most effective for studying PCDH10 methylation status?

PCDH10 promoter methylation represents a key mechanism for its downregulation in cancers. The following methodological approaches provide comprehensive methylation analysis:

• Bisulfite sequencing:

  • Extract genomic DNA from tissues or cell lines

  • Perform bisulfite conversion using commercial kits (e.g., EpiTect Bisulfite Kit)

  • Design primers specific to bisulfite-converted DNA

  • Sequence PCR products to identify methylated cytosines

  • Analyze methylation patterns across multiple CpG sites

• Methylation-specific PCR (MSP):

  • Design primer sets specific for methylated and unmethylated sequences

  • Perform parallel PCR reactions with both primer sets

  • Compare band intensities to determine methylation status

  • Include positive controls (universally methylated DNA) and negative controls

• Quantitative MSP (qMSP):

  • Utilize real-time PCR for quantitative assessment of methylation

  • Calculate percent methylation using standard curves

  • Normalize to reference genes to account for DNA quantity variations

• Methylation treatment studies:

  • Treat cells with 5-aza-2'-deoxycytidine to inhibit DNA methylation

  • Monitor PCDH10 expression changes using RT-qPCR and Western blot

  • Correlate expression recovery with methylation status changes

This comprehensive approach allows researchers to establish direct relationships between PCDH10 promoter methylation and expression levels in experimental models.

How should researchers interpret conflicting results in PCDH10 functional studies?

When encountering conflicting results in PCDH10 functional studies, researchers should systematically evaluate:

• Experimental model differences:

  • Cell line variations: Different cell types may have distinct baseline expression levels and regulatory mechanisms for PCDH10

  • In vitro vs. in vivo: Results from cell culture may differ from animal models due to microenvironmental factors

  • Primary cells vs. established lines: Immortalized cell lines may have altered signaling pathways affecting PCDH10 function

• Technical variations:

  • Antibody specificity: Different antibodies may recognize distinct epitopes or isoforms

  • Expression level assessment methods: Protein vs. mRNA quantification can yield divergent results

  • Functional assay sensitivity: Different proliferation or apoptosis assays have varying detection thresholds

• Context-dependent functions:

  • Tissue specificity: PCDH10 may have different roles in brain tissue versus cancerous tissues

  • Pathway interactions: The PI3K/Akt pathway is subject to complex regulation that may differ between experimental systems

  • Compensatory mechanisms: Other protocadherin family members may compensate for PCDH10 loss in certain contexts

• Resolution approaches:

  • Perform side-by-side comparisons using standardized protocols

  • Utilize multiple antibodies targeting different epitopes

  • Employ complementary functional assays to corroborate findings

  • Conduct experiments in multiple cell lines to determine context specificity

How does PCDH10 function in hepatocellular carcinoma progression?

PCDH10 plays a significant tumor-suppressive role in hepatocellular carcinoma (HCC) through several key mechanisms:

• Expression pattern: PCDH10 expression is significantly downregulated in HCC cells (HepG2, HuH7, HuH1, and SNU387) compared to normal liver cells (L02), suggesting its potential role as a tumor suppressor .

• Growth inhibition: Upregulation of PCDH10 through transfection with pcDNA3.1-PCDH10 plasmid significantly inhibits cell proliferation as demonstrated by CCK-8 assays. This inhibitory effect is observed at 24, 48, and 72 hours post-transfection .

• Colony formation suppression: PCDH10 overexpression results in a marked decrease in both the number and size of colonies in HCC cell lines, further confirming its growth-inhibitory properties .

• Cell cycle regulation: Flow cytometric analysis reveals that PCDH10 arrests the cell cycle at the G1 phase, with a corresponding decrease in cells in the G2 phase. This cell cycle arrest contributes to its anti-proliferative effects .

• Apoptosis induction: PCDH10 promotes apoptosis in HCC cells, likely through modulation of apoptotic pathway components like caspase-3, Bax, and Bcl-2 .

• Signaling pathway inhibition: Mechanistically, PCDH10 inhibits the PI3K/Akt signaling pathway, a critical mediator of cell survival and proliferation. Co-immunoprecipitation experiments demonstrate that PCDH10 physically interacts with the p85 subunit of PI3K, potentially explaining its inhibitory effect on this pathway .

This multifaceted tumor-suppressive function suggests PCDH10 could serve as both a biomarker and potential therapeutic target in HCC .

What experimental protocols are recommended for studying PCDH10's role in neurological disorders?

For investigating PCDH10's functions in neurological disorders, researchers should implement the following experimental protocols:

• Expression analysis in neuronal tissues:

  • RT-qPCR: Quantify PCDH10 mRNA expression in different brain regions

  • Western blot: Assess protein levels using neural-specific antibodies

  • Immunohistochemistry: Visualize expression patterns in brain sections

  • Single-cell RNA sequencing: Identify cell type-specific expression profiles

• Synaptic function assessment:

  • Electrophysiology: Evaluate synaptic transmission in PCDH10-deficient neurons

  • Synaptic protein analysis: Quantify synaptic markers (PSD95, synaptophysin) via Western blot

  • Dendritic spine morphology: Assess using confocal microscopy after Golgi staining

  • Live imaging: Monitor synaptic activity using fluorescent calcium indicators

• Axon guidance and neuronal migration studies:

  • In vitro stripe assays: Assess axon guidance preferences

  • Time-lapse imaging: Track neuronal migration in PCDH10-manipulated cultures

  • Axon outgrowth assays: Measure neurite length and branching

• Behavioral assessment in animal models:

  • PCDH10 knockout or knockdown models: Generate using CRISPR/Cas9 or conditional approaches

  • Social interaction tests: Evaluate autism-relevant behaviors

  • Learning and memory paradigms: Assess cognitive functions

  • Anxiety and depression measures: Evaluate emotional regulation

• Protein interaction studies:

  • Co-immunoprecipitation: Identify PCDH10-interacting proteins in neuronal contexts

  • Proximity ligation assay: Visualize protein-protein interactions in situ

  • Crosslinking mass spectrometry: Map interaction domains

These protocols should be implemented with appropriate controls and validated antibodies specific to neuronal PCDH10 isoforms .

How can researchers optimize PCDH10 antibody-based detection in heterogeneous tissue samples?

Optimizing PCDH10 antibody-based detection in heterogeneous tissues requires addressing several technical challenges:

• Antibody selection strategies:

  • Validate multiple antibodies recognizing different epitopes

  • Confirm specificity using PCDH10 knockout/knockdown controls

  • Consider using a combination of monoclonal and polyclonal antibodies for confirmation

  • Select antibodies with demonstrated reactivity in the tissue of interest

• Signal enhancement techniques:

  • For IHC applications: Implement heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

  • For IF applications: Use tyramide signal amplification (TSA) to boost signal

  • For Western blot: Employ enhanced chemiluminescence detection systems

  • Consider using biotin-streptavidin amplification methods for weak signals

• Background reduction methods:

  • Optimize blocking conditions (5% BSA or 5% milk, consider adding 0.1-0.3% Triton X-100)

  • Increase washing duration and frequency (at least 3 x 10 minutes per wash)

  • Pre-absorb antibodies with tissues lacking PCDH10 expression

  • Include appropriate isotype controls

• Multiplexed detection approaches:

  • Combine PCDH10 staining with cell-type-specific markers

  • Utilize sequential multiplexed immunofluorescence with spectral unmixing

  • Consider implementing multiplex immunohistochemistry with sequential antibody stripping and reprobing

• Quantification strategies:

  • Employ digital image analysis with machine learning algorithms for tissue segmentation

  • Implement cell-by-cell analysis rather than whole-tissue measurements

  • Establish signal intensity thresholds based on positive and negative controls

  • Report results as H-scores or percentage of positive cells within specific regions

These optimization approaches enable reliable detection of PCDH10 in complex tissues such as brain sections or heterogeneous tumor samples.

What strategies can resolve non-specific binding issues with PCDH10 antibodies?

Non-specific binding is a common challenge with PCDH10 antibodies due to sequence homology with other protocadherin family members. Researchers can implement these strategies to improve specificity:

• Antibody validation and optimization:

  • Test antibodies on PCDH10 knockout/knockdown controls

  • Titrate antibody concentrations to determine optimal working dilutions

  • Compare multiple antibodies targeting different epitopes

  • Validate with recombinant PCDH10 protein as a positive control

• Blocking optimizations:

  • Extend blocking time to 2 hours at room temperature

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Add 1-5% of the species serum matching the secondary antibody host

  • Include 0.1-0.3% Triton X-100 in blocking buffer for membrane permeabilization

• Washing modifications:

  • Increase wash buffer stringency (add 0.1-0.5% Tween-20)

  • Extend washing steps (5-6 washes of 10 minutes each)

  • Implement high-salt wash steps (up to 500 mM NaCl) for removing weakly bound antibodies

  • Consider using ultrasonic washers for more effective removal of non-specific binding

• Pre-absorption techniques:

  • Pre-incubate antibodies with related protocadherin family proteins

  • Use tissue/cell lysates from PCDH10-negative samples for pre-absorption

  • Implement competitive blocking with immunizing peptides

  • Consider dual epitope detection requirements for signal validation

These approaches significantly reduce background and increase confidence in experimental results when working with PCDH10 antibodies.

How can researchers design co-immunoprecipitation experiments to study PCDH10 protein interactions?

Designing effective co-immunoprecipitation (Co-IP) experiments for PCDH10 protein interactions requires careful planning and execution:

• Sample preparation optimization:

  • Transfect cells with plasmid pcDNA3.1-PCDH10 for overexpression studies

  • Harvest cells 48-72 hours post-transfection

  • Use gentle lysis buffers containing 1% NP-40 or 0.5% Triton X-100 with protease and phosphatase inhibitors

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Antibody selection and validation:

  • Use PCDH10 antibodies proven effective in immunoprecipitation applications

  • Include control antibodies (normal IgG from the same species)

  • For known interactions like PI3K p85, use specific antibodies targeting these proteins

  • Consider epitope-tagged PCDH10 constructs (FLAG, HA, Myc) for reliable Co-IP using tag antibodies

• Experimental conditions optimization:

  • Perform antibody incubation overnight at 4°C with gentle rotation

  • Optimize antibody-to-lysate ratios (typically 2-5 μg antibody per 500 μg protein)

  • Include detergent titration experiments to preserve relevant interactions

  • Consider crosslinking approaches for transient interactions

• Controls and validation:

  • Include "no antibody" controls

  • Use reciprocal Co-IP (pull down with partner antibody and probe for PCDH10)

  • Perform input controls (typically 5-10% of starting material)

  • Validate interactions with alternative methods (proximity ligation assay, FRET)

• Detection and analysis:

  • Use clean detection antibodies from different host species than IP antibodies

  • Implement Western blot with reduced secondary antibody cross-reactivity

  • Consider mass spectrometry for unbiased interaction partner identification

  • Validate novel interactions with functional studies

This methodical approach has proven effective in identifying PCDH10's interaction with the PI3K p85 subunit, revealing its mechanism of action in the PI3K/Akt signaling pathway inhibition .

What are the current methodological limitations in PCDH10 research and potential solutions?

Current methodological limitations in PCDH10 research and their potential solutions include:

• Antibody specificity challenges:

  • Limitation: Cross-reactivity with other protocadherin family members

  • Solutions:

    • Develop isoform-specific antibodies using unique epitopes

    • Implement CRISPR/Cas9 knockout controls for antibody validation

    • Combine multiple detection methods for confirmation

• Functional redundancy within the protocadherin family:

  • Limitation: Compensatory mechanisms mask phenotypes in single-gene studies

  • Solutions:

    • Design combinatorial knockdown/knockout approaches

    • Implement dominant-negative constructs targeting shared pathways

    • Utilize conditional and tissue-specific genetic modifications

• Context-dependent functions:

  • Limitation: PCDH10's role varies between tissues and developmental stages

  • Solutions:

    • Develop temporally controlled expression systems

    • Create tissue-specific reporter lines to track expression

    • Implement single-cell approaches to capture cellular heterogeneity

• Limited in vivo models:

  • Limitation: Most studies rely on cell lines rather than physiological systems

  • Solutions:

    • Develop conditional knockout mouse models

    • Utilize organoid systems for 3D tissue architecture

    • Implement patient-derived xenografts for cancer studies

• Detection of post-translational modifications:

  • Limitation: Glycosylation affects antibody recognition and protein function

  • Solutions:

    • Generate modification-specific antibodies

    • Implement mass spectrometry approaches for PTM mapping

    • Use enzymatic deglycosylation controls in parallel experiments

Addressing these limitations through methodological innovations will significantly advance our understanding of PCDH10's diverse biological functions in development and disease.

What emerging technologies hold promise for advancing PCDH10 antibody-based research?

Several cutting-edge technologies are poised to revolutionize PCDH10 antibody-based research:

• Single-cell proteomics:

  • Mass cytometry (CyTOF) for simultaneous detection of PCDH10 and dozens of other proteins at single-cell resolution

  • Multiplexed ion beam imaging (MIBI) for spatial mapping of PCDH10 in tissue contexts

  • Single-cell Western blotting for quantitative protein analysis in individual cells

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM, PALM) for nanoscale localization of PCDH10 at synapses

  • Expansion microscopy for physical magnification of subcellular structures

  • Light-sheet microscopy for rapid 3D imaging of PCDH10 in developing tissues

• Proximity labeling technologies:

  • BioID or TurboID fusions with PCDH10 for identifying proximal interacting proteins

  • APEX2-based proximity labeling for subcellular interactome mapping

  • Split-BioID for detecting conditional protein interactions

• Antibody engineering approaches:

  • Nanobodies with improved tissue penetration and specificity

  • Bispecific antibodies targeting PCDH10 and interacting partners simultaneously

  • Recombinant antibody fragments with enhanced epitope access in complex tissues

• Real-time detection systems:

  • FRET-based biosensors for monitoring PCDH10 conformational changes

  • Split fluorescent protein complementation for visualizing interactions in living cells

  • Optogenetic tools coupled with PCDH10 for spatiotemporal functional control

These emerging technologies will enable unprecedented insights into PCDH10's localization, interactions, and functions across different biological contexts.

How can PCDH10 antibodies be utilized to develop potential diagnostic or therapeutic approaches?

PCDH10 antibodies hold significant potential for diagnostic and therapeutic applications:

• Diagnostic applications:

  • Immunohistochemical detection of PCDH10 in tumor biopsies as a prognostic biomarker

  • Development of antibody-based ELISA assays for detecting PCDH10 methylation status

  • Multiplexed antibody panels combining PCDH10 with other cancer biomarkers

  • Liquid biopsy approaches detecting PCDH10-expressing circulating tumor cells

• Theranostic approaches:

  • PCDH10 antibody conjugates for simultaneous imaging and treatment

  • Antibody-drug conjugates targeting cells with aberrant PCDH10 expression

  • Development of bispecific antibodies linking PCDH10 to immune effector molecules

  • Nanoparticle-conjugated antibodies for targeted drug delivery

• Therapeutic strategies:

  • Epigenetic modifiers to restore PCDH10 expression in cancers with promoter hypermethylation

  • Small molecule screening for compounds that mimic PCDH10's tumor-suppressive effects

  • Development of proteolysis-targeting chimeras (PROTACs) for selective protein degradation

  • CRISPR-based approaches for epigenetic editing of the PCDH10 promoter

• Clinical research applications:

  • Patient stratification based on PCDH10 expression/methylation patterns

  • Response monitoring using PCDH10 as a pharmacodynamic biomarker

  • Combination therapies targeting PCDH10-regulated pathways

  • Development of companion diagnostics for PI3K/Akt pathway inhibitors

The development of these approaches requires rigorous validation of antibody specificity and correlation of PCDH10 status with clinical outcomes across diverse patient populations.

What are the key considerations for interpreting PCDH10 antibody-based research results?

When interpreting PCDH10 antibody-based research results, researchers should consider:

• Antibody validation status: Results should be interpreted in light of the antibody's validation level, including specificity testing against PCDH10 knockout controls and cross-reactivity assessment with related protocadherins .

• Experimental context: Consider the biological context, including cell/tissue type, developmental stage, and disease state, as PCDH10 functions can vary significantly across different contexts .

• Detection method limitations: Each detection method (Western blot, IHC, IF) has inherent limitations that may influence result interpretation. Multiple methods should ideally confirm key findings .

• Expression level considerations: Due to generally low endogenous expression, interpretation should account for detection sensitivity limits and potential artifacts from overexpression systems .

• Pathway interaction complexity: PCDH10's role in signaling pathways like PI3K/Akt involves complex interactions that may be influenced by other cellular factors. Consider the broader signaling context when interpreting functional results .

• Isoform specificity: Results should specify which PCDH10 isoform was detected, as different isoforms may have distinct functions .

• Translational relevance: When interpreting results for potential clinical applications, consider how in vitro findings might translate to in vivo contexts and eventually to clinical settings .