PCDH15 Antibody

What is PCDH15 and why is it significant for antibody-based research?

PCDH15 (protocadherin-15) is a calcium-dependent cell-adhesion protein essential for maintaining normal retinal and cochlear function. In humans, the canonical protein has a reported length of 1955 amino acid residues and a mass of 216.1 kDa, with its subcellular localization primarily in the cell membrane . The protein is particularly abundant in sensory cells of the inner ear and retina, highlighting its crucial role in sensory functions . Up to five different isoforms have been reported for this protein, adding complexity to experimental design considerations when selecting antibodies . PCDH15 is widely expressed in various tissues, including brain, lung, kidney, spleen, and testis, though protein expression analysis shows discernible signals predominantly in the brain . The PCDH15 gene has been associated with Usher syndrome 1F, making it an important target for studying sensory disorders, while recent evidence also implicates it in psychiatric conditions such as bipolar disorder and schizophrenia .

What types of PCDH15 antibodies are available for research applications?

Researchers have access to a diverse range of PCDH15 antibodies spanning multiple formats and production technologies. Polyclonal antibodies, such as those offered by Boster Bio and Aviva Systems Biology, provide broad epitope recognition, which can be advantageous for detecting various protein forms or partially denatured targets . Monoclonal antibodies, including recombinant options like Abcam's EPR19600 clone, offer high specificity for particular epitopes, enabling consistent results across experiments . Multiple conjugation options are available to suit various experimental needs, including unconjugated antibodies for standard applications and fluorescent conjugates (Cy3, Dylight488) for advanced imaging techniques . Antibodies targeting different regions of PCDH15 (N-terminal, C-terminal, or internal domains) allow researchers to investigate specific protein domains or isoforms with greater precision . The selection spans different host species including rabbit and mouse, which provides flexibility when designing multi-labeling experiments or when considering potential cross-reactivity issues in the experimental system .

What are the validated applications for PCDH15 antibodies?

PCDH15 antibodies have been validated across numerous experimental techniques, providing researchers with versatile tools for protein detection. Western blotting applications have confirmed PCDH15 detection at approximately 190-216 kDa, depending on the specific isoform and post-translational modifications present in the sample . Immunohistochemistry on both paraffin-embedded (IHC-P) and frozen sections (IHC-F) has been validated, with successful demonstration of PCDH15 expression in testis tissue and various neural structures . Flow cytometry applications have been established, allowing quantitative analysis of PCDH15 expression in cell populations, particularly useful for studies involving neural progenitors or differentiated neurons . Immunocytochemistry (ICC) permits subcellular localization studies, revealing PCDH15's distribution patterns within individual cells, which is crucial for understanding its functional roles at cell membranes and potential synaptic locations . Enzyme-linked immunosorbent assay (ELISA) applications provide quantitative analysis options for PCDH15 detection in various sample types, offering researchers a method for high-throughput screening or precise quantification .

Which species do PCDH15 antibodies typically recognize and what are the cross-reactivity considerations?

PCDH15 antibodies exhibit varying species reactivity profiles that researchers must consider when designing experiments across evolutionary models. Many commercially available antibodies demonstrate reactivity with human PCDH15, making them suitable for clinical research and studies using human cell lines or tissue samples . Rodent reactivity (mouse and rat) is common among PCDH15 antibodies, facilitating research in these prevalent laboratory animal models that are frequently used in neuroscience and sensory system studies . Some antibodies display broader cross-reactivity profiles extending to additional species such as rabbit, bovine, dog, and guinea pig, which enhances their utility in comparative studies or when working with specialized animal models . The evolutionary conservation of PCDH15 across species is reflected in reports of orthologs in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken, though researchers should verify actual reactivity rather than assuming cross-reactivity based on sequence homology alone . When selecting antibodies for cross-species applications, researchers should prioritize products where the immunogen sequence is identical or highly conserved between the target species, as exemplified by antibodies raised against C-terminal peptides that share identity between human, mouse, and rat PCDH15 .

How do different PCDH15 isoforms affect antibody selection and experimental design?

PCDH15 exists in up to five different isoforms, necessitating careful consideration of antibody selection to ensure detection of the relevant protein variants. Researchers must determine which specific isoforms are expressed in their experimental system before selecting antibodies, as expression patterns can vary significantly across tissues and developmental stages . The choice between antibodies targeting conserved versus isoform-specific epitopes depends on whether the research question requires detection of all PCDH15 variants or discrimination between specific isoforms . Developmental studies should particularly consider isoform dynamics, as PCDH15 protein presence varies throughout developmental stages with a notable peak during the initial three days after birth in mouse models, indicating potential shifts in isoform prevalence during maturation . Western blotting with isoform-aware antibodies can help researchers establish a profile of PCDH15 variants present in their experimental system, with expected molecular weights ranging from approximately 190-216 kDa depending on the specific isoform and post-translational modifications . For studies focusing on PCDH15's role in specialized structures like stereocilia or retinal cells, researchers should prioritize antibodies validated to detect the specific isoforms known to function in these contexts, potentially using epitopes within functional domains such as the cadherin or transmembrane regions .

What are the methodological considerations for detecting PCDH15 in neural tissues?

Detection of PCDH15 in neural tissues requires optimization of several methodological parameters to account for the protein's distinctive expression patterns and the complexity of neural structures. Fixation protocols significantly impact PCDH15 immunodetection in brain tissues, with paraformaldehyde (4%) being commonly employed while ensuring fixation times are optimized to prevent epitope masking while maintaining tissue architecture . Antigen retrieval methods should be carefully selected based on the specific antibody and neural region being examined, with citrate buffer (pH 6.0) heat-induced epitope retrieval often proving effective for PCDH15 detection in paraffin-embedded neural samples . Background autofluorescence, particularly prominent in neural tissues containing lipofuscin, necessitates appropriate blocking steps and potentially specialized treatments such as Sudan Black B to reduce non-specific signals that might interfere with specific PCDH15 detection . When studying PCDH15's regional distribution, researchers should consider that expression varies across brain regions, with documented presence in the cortex, midbrain, cerebellum, and hippocampus, requiring experimental designs that account for this heterogeneity . For developmental studies, it's essential to note that PCDH15 expression in primary cultured neurons increases as neurons mature, suggesting that timing of analysis is critical for accurate characterization of the protein's role in neural development and function .

How can PCDH15 antibodies contribute to psychiatric disorder research?

Recent genomic studies have implicated PCDH15 in psychiatric disorders, creating new research avenues that require specialized antibody-based approaches. Copy number variants (CNVs) affecting PCDH15 have been identified in patients with bipolar disorder, schizophrenia, and autism spectrum disorder, making antibody-based protein quantification an important complementary approach to genetic studies for understanding pathogenic mechanisms . Researchers investigating PCDH15's role in psychiatric conditions should consider immunohistochemical co-localization studies with established psychiatric disorder markers to identify potentially affected neural circuits and cell populations expressing the protein . Cell-type specific analysis is particularly important as PCDH15 is highly expressed in human oligodendrocyte progenitor cells and influences white matter development and myelination, suggesting potential mechanistic links to psychiatric disorders through myelin abnormalities . Animal models with PCDH15 deficiency, such as the Pcdh15-deletion mice described in recent literature, provide valuable systems for antibody-based studies correlating protein expression with behavioral phenotypes relevant to psychiatric disorders . Importantly, researchers should note that some spontaneously occurring Pcdh15-deletion mice exhibit retinal and inner ear structural abnormalities that could confound behavioral studies, necessitating careful characterization of these sensory functions when interpreting results related to psychiatric phenotypes .

What controls should be included when validating PCDH15 antibody specificity?

Rigorous validation of PCDH15 antibody specificity requires implementation of multiple control strategies to ensure experimental results accurately reflect true protein expression. Positive tissue controls should include samples known to express PCDH15, such as brain, testis, and inner ear tissues, while negative controls might include tissues with minimal expression or samples from Pcdh15-null models when available . Genetic knockout or knockdown models provide the gold standard for antibody validation, with Pcdh15-null mice showing barely detectable PCDH15 expression levels compared to wild-type, while heterozygous models (Pcdh15-het) exhibit approximately 31% of wild-type expression, offering a spectrum of expression levels for validation . Peptide competition assays, where the antibody is pre-incubated with the immunizing peptide, should eliminate specific staining while non-specific binding remains, providing a powerful method to distinguish true from false signals, particularly relevant for PCDH15 due to its multiple isoforms and potential cross-reactivity with related protocadherins . Western blot analysis should demonstrate detection at the expected molecular weight (approximately 190-216 kDa for full-length PCDH15), with additional bands potentially representing isoforms or post-translationally modified variants . For developmental studies, researchers should verify that antibody detection patterns align with known expression profiles, such as PCDH15's peak expression during the initial three days after birth and its increasing expression as cultured neurons mature .

What are the recommended protocols for PCDH15 Western blot analysis?

Optimizing Western blot protocols for PCDH15 detection requires attention to several technical parameters due to its high molecular weight and variable expression levels. Sample preparation should include efficient extraction using buffers containing protease inhibitors to prevent degradation, with particular attention to membrane protein extraction techniques given PCDH15's cell membrane localization . Gel concentration requires careful consideration, with lower percentage gels (6-8% acrylamide) recommended to facilitate efficient separation and transfer of the high molecular weight PCDH15 protein (190-216 kDa) . Transfer conditions should be optimized for large proteins, potentially using lower current for extended periods or specialized transfer systems designed for high molecular weight proteins to ensure complete transfer to the membrane . Blocking solutions may require optimization, with 5% non-fat dry milk or BSA in TBST commonly effective, though researchers should test both to determine which provides optimal signal-to-noise ratio for their specific PCDH15 antibody . Primary antibody dilutions typically range from 1:500 to 1:1000 for PCDH15 antibodies, but optimal concentration should be determined empirically for each application and antibody lot, with incubation preferably performed overnight at 4°C to maximize specific binding .

How should researchers optimize immunohistochemistry protocols for PCDH15 detection?

Successful immunohistochemical detection of PCDH15 requires careful optimization of multiple experimental parameters to achieve specific staining with minimal background. Fixation methods significantly impact PCDH15 epitope preservation, with 4% paraformaldehyde commonly used for neural tissues, though fixation duration should be optimized to prevent overfixation that might mask epitopes while ensuring adequate tissue preservation . Antigen retrieval is often essential, with heat-induced epitope retrieval using citrate buffer (pH 6.0) frequently effective for paraffin sections, though EDTA-based buffers may prove superior for certain antibodies and should be tested empirically . Antibody dilution ranges typically span from 1:100 to 1:500 for immunohistochemistry applications, as exemplified by the successful staining of human testis tissue with ab230346 at 1:100 dilution, though optimal concentration must be determined for each tissue type and antibody . Blocking protocols should address both endogenous peroxidase activity (if using HRP-based detection) and non-specific binding sites, with 3% hydrogen peroxide followed by serum from the species of the secondary antibody (typically 5-10%) providing effective blocking for most applications . Detection systems should be selected based on expression level expectations, with standard ABC or polymer-based systems suitable for moderate to high expression, while tyramide signal amplification might be necessary for detecting low abundance or partially masked PCDH15 epitopes .

What techniques are recommended for co-localization studies involving PCDH15?

Co-localization studies examining PCDH15's relationship with other proteins require meticulous attention to antibody compatibility and imaging parameters. Antibody selection should prioritize primary antibodies raised in different host species (such as rabbit anti-PCDH15 paired with mouse antibodies against other targets) to enable simultaneous detection without cross-reactivity between secondary antibodies . Sequential immunostaining protocols may be necessary when using multiple antibodies from the same host species, employing complete blocking steps between rounds of primary and secondary antibody application to prevent cross-detection . Fluorophore selection should consider spectral overlap and the specific characteristics of the imaging system, with widely separated emission spectra (e.g., combining Cy3-conjugated anti-PCDH15 with Alexa 488 or Dylight 647 conjugates for other targets) minimizing bleed-through during image acquisition . Particularly valuable co-localization targets include CDH23, which interacts with PCDH15 via their extracellular domains and has been linked to schizophrenia and attention deficit hyperactivity disorder, potentially revealing functional protein complexes relevant to neuropsychiatric conditions . For developmental studies, co-localization with stage-specific neural markers can reveal temporal relationships between PCDH15 expression and key developmental processes, especially given that PCDH15 protein increases as neurons mature and peaks during early postnatal development .

How can researchers quantify PCDH15 expression levels accurately?

Accurate quantification of PCDH15 expression requires carefully designed protocols that account for technical variability while enabling meaningful biological comparisons. Western blot quantification should employ housekeeping protein normalization appropriate for the experimental context, with membrane protein loading controls (such as Na+/K+ ATPase) potentially providing more relevant normalization than cytoskeletal or cytoplasmic standards given PCDH15's membrane localization . For immunohistochemical quantification, researchers should establish standardized image acquisition parameters and analysis workflows, potentially utilizing automated counting of positive cells or intensity measurement within defined regions of interest while maintaining consistent thresholding criteria across all experimental groups . When comparing expression levels between wild-type and modified systems, calibration against established models can provide valuable reference points, as exemplified in studies showing PCDH15 expression in Pcdh15-heterozygous mice at approximately 31% of wild-type levels . Flow cytometry offers quantitative single-cell analysis of PCDH15 expression, particularly valuable for heterogeneous neural populations, with careful gating strategies required to distinguish specific populations and appropriate isotype controls to establish background fluorescence levels . For developmental expression studies, consistent sampling timepoints are critical given that PCDH15 protein levels peak during the initial three days after birth and increase as cultured neurons mature, necessitating precise timing of sample collection to capture dynamic expression changes .

How can researchers address weak or inconsistent PCDH15 detection?

When facing challenges with PCDH15 detection, systematic troubleshooting approaches can help identify and resolve technical issues affecting signal quality. Sample preparation optimization is often critical, particularly ensuring complete solubilization of membrane-bound PCDH15 using appropriate detergents (such as NP-40 or Triton X-100) in lysis buffers, potentially with longer extraction times or alternative extraction methods specifically designed for membrane proteins . For Western blotting applications, extended transfer times or specialized transfer conditions for high molecular weight proteins may improve detection of the full-length 190-216 kDa PCDH15 protein, as incomplete transfer is a common cause of weak signals with large proteins . Antigen retrieval protocols should be systematically tested when working with fixed tissues, comparing heat-induced retrieval methods using different buffers (citrate, EDTA, or Tris-based) and pH conditions to identify optimal parameters for exposing PCDH15 epitopes that may be masked during fixation . When signal remains weak despite optimization, signal amplification systems such as biotin-streptavidin enhancement, tyramide signal amplification, or polymer-based detection systems may significantly improve sensitivity while maintaining specificity, particularly valuable for tissues with lower PCDH15 expression levels . For developmental studies, researchers should consider that PCDH15 expression varies significantly across developmental stages, with peak expression in early postnatal periods in mice, suggesting that timing of analysis is critical for successful detection .

What approaches can help distinguish between specific and non-specific signals?

Distinguishing genuine PCDH15 signals from non-specific background requires implementation of rigorous validation strategies and appropriate controls. Peptide competition assays provide compelling evidence of antibody specificity, as pre-incubation of the PCDH15 antibody with its immunizing peptide should eliminate specific staining while leaving non-specific signals unchanged, allowing researchers to identify true PCDH15 detection . Genetic models offer powerful validation tools, with comparison of signals between wild-type, heterozygous (showing approximately 31% of wild-type PCDH15 levels), and knockout tissues (where PCDH15 is barely detectable) enabling researchers to confirm that signal intensity correlates with expected expression levels . Technical replication using antibodies targeting different PCDH15 epitopes can strengthen confidence in detection specificity, as concordant results from antibodies recognizing distinct protein regions (such as N-terminal versus C-terminal domains) provide strong evidence for authentic PCDH15 detection . For Western blotting applications, molecular weight verification is essential, with specific PCDH15 detection expected at approximately 190-216 kDa, though researchers should note that post-translational modifications (particularly glycosylation) and different isoforms may cause some variation in apparent molecular weight . When evaluating immunohistochemical staining patterns, comparison with published PCDH15 expression data can help verify specificity, with expected expression in specific tissues (brain, inner ear, retina, testis) and subcellular localization (cell membrane) serving as important reference points .

What are common pitfalls in PCDH15 immunohistochemistry and how can they be avoided?

Immunohistochemical detection of PCDH15 presents several common challenges that require specific technical approaches to overcome. Overfixation frequently compromises PCDH15 epitope accessibility, particularly in neural tissues where extended fixation can mask antibody binding sites; researchers should optimize fixation duration (typically 24-48 hours for adult brain samples) and consider post-fixation treatments such as formic acid for improved epitope exposure . Autofluorescence represents a significant challenge in tissues like brain and retina where PCDH15 is expressed, potentially confounding specific signals; treatments such as Sudan Black B (0.1-0.3%) or specialized autofluorescence quenching kits can significantly reduce this interference when performing fluorescent immunohistochemistry . Edge artifacts are common when staining for membrane proteins like PCDH15, creating false positive signals at tissue boundaries; these can be minimized through careful tissue handling, appropriate blocking protocols, and critical evaluation comparing staining patterns at tissue edges versus internal regions . Non-specific nuclear staining sometimes occurs with PCDH15 antibodies despite the protein's membrane localization; this can be reduced by extensive blocking with both serum and protein blockers (such as 5% normal serum combined with 2% BSA) and careful optimization of antibody concentration to minimize non-specific binding while maintaining specific detection . For quantitative studies, inconsistent staining across sections can introduce significant variability; this can be addressed through batch processing of all experimental groups simultaneously with consistent reagents and timing, or by including standard reference samples in each batch to enable cross-batch normalization .

How are PCDH15 antibodies contributing to psychiatric disorder research?

Recent genomic studies have established connections between PCDH15 variants and psychiatric conditions, creating new avenues for antibody-based investigation of underlying mechanisms. Copy number variants (CNVs) affecting PCDH15 have been identified in patients with bipolar disorder, schizophrenia, and autism spectrum disorder, enabling researchers to use antibodies to examine how these genetic variations translate to altered protein expression patterns in affected neural circuits . Antibody-based studies in animal models with PCDH15 deficiency provide platforms for correlating protein expression with behavioral phenotypes relevant to psychiatric disorders, though researchers must carefully account for potential sensory abnormalities that might confound interpretations of behavioral assessments . The interaction between PCDH15 and cadherin23 (CDH23) presents a particularly interesting target for psychiatric research, as CDH23 has been independently linked to schizophrenia and attention deficit hyperactivity disorder, suggesting that antibody-based co-localization studies might reveal disrupted protein complexes relevant to disease pathophysiology . PCDH15's high expression in oligodendrocyte progenitor cells and its influence on white matter development and myelination offers an intriguing connection to psychiatric disorders, which frequently feature white matter abnormalities; immunohistochemical characterization of PCDH15 in oligodendrocyte lineage cells may therefore illuminate novel pathogenic mechanisms . Moving forward, combining PCDH15 antibody-based protein quantification with functional assessments in relevant neural circuits will be essential for translating genetic associations into mechanistic understanding of how PCDH15 alterations contribute to psychiatric disorder pathophysiology .

What is the significance of PCDH15 in sensory system research?

PCDH15 plays fundamental roles in sensory systems, making antibodies against this protein invaluable tools for investigating normal function and pathological conditions affecting vision and hearing. In the inner ear, PCDH15 is critical for proper stereocilia function in hair cells, with antibody-based localization studies revealing its precise distribution within these specialized structures and how mutations associated with Usher syndrome 1F disrupt this organization . Retinal expression of PCDH15 highlights its importance in vision, with immunohistochemical studies using specific antibodies helping to characterize its distribution across different retinal layers and cell types, providing insights into how mutations lead to the visual impairments characteristic of Usher syndrome . Developmental studies benefit from PCDH15 antibodies that can track expression changes across critical periods of sensory system formation, particularly given the protein's dynamic expression pattern that peaks during early postnatal development in mice . For researchers investigating Usher syndrome models, antibodies enable assessment of how specific mutations affect PCDH15 protein levels, subcellular localization, and interactions with partner proteins, potentially informing therapeutic approaches aimed at correcting or compensating for these molecular defects . The evolutionary conservation of PCDH15 across species facilitates comparative studies using antibodies reactive with orthologs in various model organisms, allowing researchers to investigate both conserved and species-specific aspects of sensory system development and function from zebrafish to mammals .

How can PCDH15 antibodies advance studies of neural development and synapse formation?

PCDH15's roles in neural differentiation and synaptogenesis create opportunities for antibody-based investigations of fundamental neurodevelopmental processes. Temporal expression pattern analysis using PCDH15 antibodies reveals that the protein increases as neurons mature in culture and peaks during early postnatal development in mice, suggesting critical developmental windows where it may influence neural circuit formation . Co-localization studies combining PCDH15 antibodies with markers for specific neural cell types and subcellular compartments can characterize its distribution across developing synapses, potentially illuminating its contributions to synaptic adhesion, specification, or maintenance . The protein's expression across diverse brain regions including the cortex, midbrain, cerebellum, and hippocampus indicates potential roles in multiple neural circuits, with antibody-based mapping enabling researchers to prioritize regions for functional studies of PCDH15's impact on circuit development and plasticity . PCDH15's influence on oligodendrocyte progenitor cell proliferation and white matter development represents a particularly promising area for antibody-based research, potentially revealing novel mechanisms through which this adhesion molecule contributes to myelination processes critical for neural function . For developmental neurotoxicology studies, PCDH15 antibodies provide tools to assess whether environmental toxins or pharmaceuticals disrupt normal expression patterns during critical developmental windows, potentially identifying molecular mechanisms underlying neurodevelopmental disorders associated with environmental exposures .

What emerging technologies are enhancing PCDH15 antibody applications in research?

Advanced technological approaches are expanding the utility of PCDH15 antibodies and enabling more sophisticated analyses of this protein's functions. Super-resolution microscopy techniques such as STORM, PALM, and structured illumination microscopy overcome the diffraction limit of conventional microscopy, allowing researchers to visualize PCDH15's precise nanoscale organization within specialized structures like stereocilia or synaptic complexes when combined with high-quality specific antibodies . Proximity ligation assays utilizing PCDH15 antibodies can detect protein-protein interactions in situ with high sensitivity, providing powerful tools for studying PCDH15's associations with binding partners like cadherin23 under various physiological or pathological conditions . Mass cytometry (CyTOF) combined with PCDH15 antibodies enables high-dimensional single-cell analysis of protein expression across heterogeneous neural populations, potentially revealing cell-type specific regulation patterns that might be missed in bulk analysis approaches . Tissue clearing techniques compatible with immunolabeling, such as CLARITY, iDISCO, and CUBIC, allow whole-organ imaging of PCDH15 distribution using specific antibodies, providing three-dimensional perspectives on expression patterns across intact neural structures . Single-molecule pull-down approaches using highly specific PCDH15 antibodies can isolate native protein complexes for proteomic analysis, potentially identifying novel interaction partners and advancing understanding of the protein's diverse functions in different cellular contexts .