PCDH17 Antibody

What is PCDH17 and why is it important in research?

Protocadherin 17 (PCDH17) is a 120-160 kDa glycoprotein belonging to the δ2-group of the protocadherin subfamily within the cadherin family of molecules. It functions primarily as a cell adhesion molecule expressed on endothelium and various epithelia, including stratified squamous epithelium and gastric columnar epithelium . PCDH17 has gained significant research interest due to its roles in multiple biological processes, including neuronal development, cell cycle regulation, cell migration inhibition, and its implications in various pathological conditions . Research has demonstrated PCDH17's involvement in cancer progression, particularly colorectal cancer sensitivity to chemotherapy, as well as its roles in neuropsychiatric conditions including major mood disorders .

What is the molecular structure and cellular localization of PCDH17?

PCDH17 is a type I transmembrane protein with a molecular weight ranging from 120-160 kDa, though the calculated molecular weight is approximately 126 kDa (1159 amino acids) . The mature human PCDH17 protein contains:

• A 690 amino acid extracellular domain (ECD) spanning residues 18-707

• Six cadherin domains within the ECD (residues 18-695)

• A 431 amino acid cytoplasmic region

• At least two splice variants involving substitutions in the cytoplasmic region

PCDH17 is primarily localized to the cell membrane, consistent with its function in cell-cell adhesion . The protein undergoes post-translational modifications, particularly glycosylation, which accounts for the discrepancy between its calculated molecular weight (126 kDa) and observed molecular weight (160-170 kDa) in Western blot analyses .

What are the recommended applications for PCDH17 antibodies?

Based on current research protocols, PCDH17 antibodies have been validated for the following applications:

It is strongly recommended that each laboratory determine optimal dilutions for their specific experimental systems, as antibody performance can vary based on sample type, detection method, and experimental conditions . When using PCDH17 antibodies in Western blot applications, researchers should expect to detect bands at approximately 120-170 kDa, depending on the sample type and specific antibody used .

What are the optimal conditions for Western blot detection of PCDH17?

For optimal Western blot detection of PCDH17, researchers should follow these methodological guidelines:

• Sample preparation: Brain tissue (particularly cerebellum or cortex) provides strong PCDH17 signal in mouse and rat models . Lysates should be prepared under reducing conditions.

• Recommended protocol:

  • Use PVDF membrane for protein transfer

  • Block with appropriate blocking buffer (typically 5% non-fat milk or BSA)

  • Primary antibody dilution: 1:500-1:1000 in blocking buffer

  • Incubate with species-appropriate HRP-conjugated secondary antibody

  • Develop using standard chemiluminescence detection

• Expected results: PCDH17 should be detected at approximately 160-170 kDa in human samples and 120-160 kDa in mouse and rat brain tissues . The specific band size may vary slightly depending on tissue type and post-translational modifications.

• Controls: Include positive controls such as mouse brain cortex or rat brain cerebellum tissues, which have been validated to express PCDH17 . Negative controls should include tissues known not to express PCDH17 or siRNA knockdown samples.

How should PCDH17 antibodies be stored and handled to maintain reactivity?

To preserve antibody performance and ensure experimental reproducibility, PCDH17 antibodies should be handled according to these storage guidelines:

• Long-term storage: Store antibodies at -20°C to -70°C. Most PCDH17 antibodies remain stable for up to 12 months from the date of receipt when stored properly .

• Working solution preparation:

  • Thaw antibodies on ice or at 2-8°C (avoid room temperature thawing)

  • Use a manual defrost freezer and avoid repeated freeze-thaw cycles, which can lead to protein denaturation and decreased activity

  • Aliquot antibodies before freezing to minimize freeze-thaw cycles

• Short-term storage:

  • Reconstituted antibodies can be stored at 2-8°C for up to 1 month under sterile conditions

  • For longer periods (up to 6 months), store reconstituted antibodies at -20°C to -70°C under sterile conditions

• Buffer conditions: Most PCDH17 antibodies are provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability . Some smaller-volume preparations may contain 0.1% BSA as a stabilizer .

How can I validate the specificity of a PCDH17 antibody in my experimental system?

Validation of antibody specificity is critical for obtaining reliable results. For PCDH17 antibodies, consider these validation approaches:

• Positive and negative controls:

  • Use tissues with known PCDH17 expression (mouse/rat brain tissues) as positive controls

  • Include tissues where PCDH17 is not expressed as negative controls

  • Compare with recombinant PCDH17 protein of known concentration

• Molecular approaches:

  • siRNA or shRNA knockdown of PCDH17 in cell lines expressing the protein should reduce or eliminate the signal

  • Overexpression systems can confirm the molecular weight of the detected protein

  • CRISPR/Cas9 knockout cells provide definitive negative controls

• Cross-reactivity assessment:

  • Test the antibody against related protocadherins to ensure specificity

  • Human PCDH17 shares 98% amino acid identity with mouse PCDH17 over amino acids 18-705, making cross-species reactivity likely but requiring validation

• Application-specific validation:

  • For Western blot, verify the band appears at the expected molecular weight (160-170 kDa)

  • For immunohistochemistry, compare staining patterns with published literature on PCDH17 expression

How can PCDH17 antibodies be used to investigate its role in cancer progression and chemotherapy sensitivity?

Research has identified PCDH17 as a potential tumor suppressor and modulator of chemotherapy sensitivity, particularly in colorectal cancer (CRC). Methodological approaches using PCDH17 antibodies include:

• Expression correlation studies:

  • Compare PCDH17 protein expression in 5-FU-sensitive versus 5-FU-resistant CRC tissues using Western blot or immunohistochemistry

  • Correlate PCDH17 expression with BECN1 (a key autophagy protein) expression, as studies have shown a positive correlation between these proteins in chemosensitive tumors

• Mechanistic studies of chemosensitivity:

  • Investigate the relationship between PCDH17 expression and apoptotic markers following 5-FU treatment

  • Examine JNK pathway activation (a key mechanism in PCDH17-induced autophagy) using phospho-specific antibodies alongside PCDH17 detection

  • Use antibodies to monitor autophagic flux in cells with manipulated PCDH17 expression

• Prognostic marker development:

  • Develop tissue microarrays to evaluate PCDH17 expression in patient cohorts

  • Correlate expression levels with treatment response and survival outcomes

  • Consider combining with other markers such as BECN1 for improved predictive power

Research has demonstrated that PCDH17 increases CRC sensitivity to 5-FU by inducing both apoptosis and JNK-dependent autophagic cell death, making it a potential prognostic marker for predicting 5-FU sensitivity in CRC patients .

What techniques can be used to study PCDH17's role in neuronal development and psychiatric disorders?

PCDH17 has been implicated in neuronal development, synaptogenesis, and psychiatric conditions, particularly major mood disorders. Advanced research approaches include:

• Structural and functional neuroimaging correlation:

  • Use PCDH17 antibodies in postmortem tissue studies to correlate protein expression with neuroimaging findings

  • Examine PCDH17 expression in specific neuronal populations, particularly in the amygdala where PCDH17 is expressed in a subset of neurons

  • Correlate PCDH17 levels with amygdala volume and function as identified in imaging studies

• Synaptic development analysis:

  • Use PCDH17 antibodies to study dendritic spine morphology and density in primary neuronal cultures

  • Combine with transfection of PCDH17 variants to study effects of overexpression on synaptic development

  • Research has shown that elevated expression of PCDH17 in primary neuronal cultures results in decreased spine density and aberrant dendritic morphology, which may underlie synaptic dysfunction in mood disorders

• Genetic-molecular correlations:

  • Examine how PCDH17 risk alleles (identified in genome-wide association studies) affect protein expression in postmortem brain samples

  • Risk alleles have been shown to predict higher PCDH17 mRNA levels, consistent with increased expression in patients with bipolar disorder compared to healthy controls

• Animal model studies:

  • Use PCDH17 antibodies to validate knockout or transgenic models

  • Compare behavioral phenotypes with protein expression patterns

  • PCDH17 knockout mice exhibit antidepressant-like phenotypes, suggesting its involvement in mood regulation

How can I distinguish between different PCDH17 isoforms in experimental systems?

PCDH17 exists in multiple isoforms due to alternative splicing, particularly in the cytoplasmic region. Advanced detection approaches include:

• Isoform-specific antibody selection:

  • Choose antibodies raised against specific regions that differ between isoforms

  • For human PCDH17, at least two splice variants have been identified that involve substitutions in the cytoplasmic region (a 14 amino acid substitution in one variant and a 25 amino acid substitution in another, both affecting residues 876-1159)

• Molecular weight discrimination:

  • Use high-resolution SDS-PAGE (6-8% gels) to separate closely migrating isoforms

  • Western blot analysis can reveal subtle differences in molecular weight between isoforms

  • Combine with isoform-specific antibodies for definitive identification

• RT-PCR validation:

  • Design primers specific to different splice junctions

  • Use RT-PCR to confirm the presence of specific mRNA isoforms

  • Correlate protein detection with mRNA expression patterns

• Mass spectrometry confirmation:

  • For definitive isoform identification, immunoprecipitate PCDH17 using the antibody

  • Analyze the precipitated proteins by mass spectrometry to identify specific peptide sequences unique to each isoform

What are common issues in Western blot detection of PCDH17 and how can they be resolved?

When working with PCDH17 antibodies in Western blot applications, researchers may encounter several technical challenges:

• High molecular weight detection issues:

  • Problem: Difficulty detecting the high molecular weight PCDH17 protein (160-170 kDa)

  • Solution: Use lower percentage gels (6-8%), extend transfer time, and employ pulsed or high-molecular-weight transfer protocols

• Multiple bands or smearing:

  • Problem: Detection of multiple bands or smearing around the expected molecular weight

  • Cause: Post-translational modifications (particularly glycosylation), protein degradation, or non-specific binding

  • Solution: Include protease inhibitors during sample preparation, optimize primary antibody dilution (1:500-1:1000), and consider using freshly prepared tissue samples

• Weak signal:

  • Problem: Low or undetectable signal despite proper sample preparation

  • Solution: Increase antibody concentration, extend primary antibody incubation time (overnight at 4°C), use more sensitive detection methods, or increase protein loading (50-100 μg of total protein is often needed for endogenous detection)

• High background:

  • Problem: Non-specific staining obscuring specific PCDH17 signal

  • Solution: Increase blocking time/concentration, optimize antibody dilution, include additional wash steps, and consider using TBS instead of PBS for antibodies sensitive to phosphate buffers

How can I optimize PCDH17 antibody performance across different sample types?

Different sample types require specific optimization strategies for PCDH17 detection:

• Brain tissue samples (highest endogenous expression):

  • Use fresh or flash-frozen samples when possible

  • For mouse/rat samples, cerebellum and cortex show reliable PCDH17 expression

  • Standard protein extraction buffers with protease inhibitors are typically sufficient

  • Expected molecular weight: 120-160 kDa in rodent brain tissues

• Cell line samples:

  • Transient transfection with PCDH17 expression constructs may be necessary for cell lines with low endogenous expression

  • For endogenous detection, neuronal cell lines typically show higher expression levels

  • Confluency can affect expression levels; standardize cell culture conditions

  • Consider using concentrated protein samples (e.g., through immunoprecipitation) for low-expressing lines

• Human patient samples:

  • Postmortem brain tissues show variable PCDH17 expression based on brain region

  • Amygdala samples are particularly relevant given PCDH17's expression in this region

  • Patient-derived samples may require more sensitive detection methods

  • Expected molecular weight: 160-170 kDa in human samples

• Sample-specific recommendations:

  • For all sample types, titrate the antibody concentration for optimal signal-to-noise ratio

  • The recommended starting dilution of 1:500-1:1000 should be adjusted based on sample type

  • Always include appropriate positive controls (e.g., brain tissue) when testing new sample types

How can I address discrepancies between mRNA expression and protein detection of PCDH17?

Researchers often encounter discrepancies between PCDH17 mRNA levels and protein detection, which can be addressed through these methodological approaches:

• Protein stability assessment:

  • PCDH17 protein may have different half-lives across tissue types

  • Use protein synthesis inhibitors (e.g., cycloheximide) to determine protein turnover rates

  • Compare with mRNA stability using actinomycin D treatment

• Post-transcriptional regulation:

  • Investigate the role of microRNAs in regulating PCDH17 translation

  • Examine RNA-binding proteins that might affect PCDH17 mRNA stability or translation

  • Consider alternative splicing events that might affect antibody binding sites

• Technical considerations:

  • Ensure the antibody epitope matches the specific PCDH17 isoform being studied

  • Use multiple antibodies targeting different regions of PCDH17

  • Combine protein detection methods (Western blot, immunoprecipitation, immunohistochemistry) to validate results

• Biological variability:

  • In psychiatric research, risk alleles have been associated with higher PCDH17 mRNA levels in postmortem brains

  • Consider genetic background when interpreting expression data

  • Examine data in the context of relevant physiological or pathological conditions

How can PCDH17 antibodies be used in the context of neurodevelopmental research?

PCDH17's role in neuronal development and synaptogenesis makes it relevant for neurodevelopmental research:

• Developmental expression profiling:

  • Use PCDH17 antibodies to track protein expression across developmental stages

  • Compare expression patterns in different brain regions, particularly the amygdala where PCDH17 is expressed in specific neuronal subsets

  • Correlate with the development of neuronal circuits and synaptic connections

• Synaptic function studies:

  • Investigate PCDH17's role in synapse formation and maintenance

  • Combine antibody labeling with synaptic markers to assess colocalization

  • Research has shown that elevated PCDH17 expression leads to decreased spine density and aberrant dendritic morphology, suggesting its importance in synaptic development

• Circuit formation analysis:

  • Use PCDH17 antibodies to identify specific neuronal populations during circuit development

  • Combine with tract-tracing studies to understand connectivity patterns

  • Assess how PCDH17 expression correlates with functional circuit establishment

• Neurodevelopmental disorder research:

  • Examine PCDH17 expression in models of neurodevelopmental disorders

  • Investigate potential disruptions in expression patterns or localization

  • Consider PCDH17 as a potential biomarker for developmental abnormalities

What is the significance of PCDH17 in cancer research beyond colorectal cancer?

While PCDH17's role in colorectal cancer has been well-studied, emerging research suggests broader implications in oncology:

• Tumor suppressor function:

  • Use PCDH17 antibodies to assess protein expression across various cancer types

  • Correlate expression levels with clinical outcomes and tumor characteristics

  • Investigate mechanisms of PCDH17 downregulation, particularly through promoter methylation

• Cell migration and invasion studies:

  • PCDH17 appears to play a role in inhibiting cell migration

  • Use antibodies to correlate protein expression with invasive capacity

  • Examine cytoskeletal changes associated with PCDH17 expression or loss

• Therapeutic response prediction:

  • Building on findings in colorectal cancer, where PCDH17 increases sensitivity to 5-FU

  • Investigate whether similar mechanisms operate in other cancer types

  • Develop predictive models based on PCDH17 expression for personalized treatment approaches

• Mechanistic pathway studies:

  • Examine PCDH17's interaction with JNK signaling across cancer types

  • Investigate relationships between PCDH17 and autophagy regulation

  • The JNK pathway has been identified as a key mediator of PCDH17-induced autophagy in colorectal cancer, which may extend to other malignancies

What are emerging technologies for studying PCDH17 function?

As research on PCDH17 continues to evolve, several cutting-edge technologies show promise for advancing our understanding:

• CRISPR/Cas9 genome editing:

  • Generate precise PCDH17 knockout or knock-in models

  • Create tagged versions of endogenous PCDH17 for localization studies

  • Introduce specific mutations corresponding to human variants

• Super-resolution microscopy:

  • Examine PCDH17 localization at synapses with nanometer precision

  • Study dynamic changes in PCDH17 distribution during neuronal development

  • Investigate co-localization with other synaptic proteins

• Single-cell approaches:

  • Combine single-cell RNA sequencing with protein detection

  • Identify cell populations with unique PCDH17 expression patterns

  • Correlate with functional characteristics of specific neuronal subtypes

• Proximity labeling techniques:

  • Use BioID or APEX2 fusion proteins to identify PCDH17 interacting partners

  • Map the PCDH17 interactome in different cellular contexts

  • Discover novel functional relationships

How might understanding PCDH17 lead to new therapeutic approaches?

The multifaceted roles of PCDH17 in development and disease suggest several therapeutic avenues:

• Cancer treatment strategies:

  • Develop approaches to restore PCDH17 expression in cancers where it is downregulated

  • Use PCDH17 expression as a biomarker for chemotherapy response, particularly for 5-FU treatment in colorectal cancer

  • Target the JNK pathway in PCDH17-expressing tumors to enhance autophagic cell death

• Neuropsychiatric disorder therapeutics:

  • Explore PCDH17 as a target for mood disorder treatments, given that knockout mice show antidepressant-like phenotypes

  • Develop modulators of PCDH17 function that could affect synaptic development

  • Consider genetic risk variants when designing personalized treatments

• Regenerative medicine applications:

  • Investigate PCDH17's potential role in guiding neuronal growth and connectivity

  • Explore its use in promoting appropriate synaptic development in regenerative approaches

  • Study its function in neuronal differentiation for stem cell-based therapies

• Biomarker development:

  • Validate PCDH17 as a prognostic or predictive biomarker for 5-FU treatment response

  • Explore its utility as a biomarker for neuropsychiatric conditions

  • Develop standardized assays for clinical implementation

What knowledge gaps remain in PCDH17 research?

Despite significant advances, several important questions about PCDH17 remain unanswered:

• Structural and functional relationships:

  • How do specific domains of PCDH17 contribute to its diverse functions?

  • What are the binding partners of PCDH17 in different cellular contexts?

  • How does the cytoplasmic domain mediate intracellular signaling?

• Regulatory mechanisms:

  • What controls PCDH17 expression during development and disease?

  • How is PCDH17 trafficking and localization regulated?

  • What post-translational modifications affect PCDH17 function?

• Cell type-specific functions:

  • Why is PCDH17 expressed in specific subsets of neurons, particularly in the amygdala?

  • Does PCDH17 function differently in different cell types?

  • How do cell type-specific partners influence PCDH17 activity?

• Translational research needs:

  • Validation of PCDH17 as a therapeutic target or biomarker across larger patient populations

  • Development of standardized assays for clinical implementation

  • Establishment of animal models that accurately recapitulate human PCDH17-related pathologies