PCDHA8 Antibody

What is PCDHA8 and what cellular functions does it perform?

PCDHA8 (Protocadherin alpha 8) is a member of the protocadherin alpha gene cluster, located on chromosome five. It belongs to the cadherin superfamily of cell adhesion molecules that form calcium-dependent homophilic complexes. PCDHA8 functions as a neural cadherin-like cell adhesion protein and plays critical roles in the establishment and maintenance of specific cell-cell connections in the brain. In neuronal tissues, PCDHA8 is found in synaptic membranes and impacts long-term potentiation (LTP). In non-neuronal tissues such as breast epithelium, it contributes to maintaining stable epithelial architecture . The protein is a type I transmembrane protein approximately 1041 amino acids in length, containing a 720-amino acid extracellular domain (ECD) with six cadherin domains and a 300-amino acid cytoplasmic region . Multiple splice variants exist, including those with deletions of amino acids 134-607 and 781-877, as well as a variant with a six amino acid substitution for residues 365-527 .

What types of PCDHA8 antibodies are available for research applications?

Several types of PCDHA8 antibodies have been developed for research applications:

These antibodies have been validated for various applications including Western Blot (WB), Immunofluorescence (IF), Immunohistochemistry (IHC), and Enzyme-Linked Immunosorbent Assay (ELISA), allowing researchers to select the appropriate antibody based on their specific experimental requirements .

How should PCDHA8 antibodies be stored to maintain optimal activity?

Proper storage of PCDHA8 antibodies is crucial for maintaining their activity and specificity. The general recommendations include:

• Short-term storage (up to three months): Store at 4°C .

• Long-term storage (up to one year): Store at –20°C .

• For reconstituted antibodies:

  • Store at 2 to 8°C under sterile conditions for up to 1 month

  • Store at -20 to -70°C under sterile conditions for up to 6 months

It is critically important to avoid repeated freeze-thaw cycles as they can lead to protein denaturation and loss of antibody activity . Aliquoting the antibody into smaller volumes before freezing is recommended to minimize freeze-thaw cycles. Some manufacturers also provide specific storage buffer recommendations that may enhance stability. Always refer to the manufacturer's instructions for product-specific storage recommendations, as formulations may vary between suppliers.

What are the recommended dilutions and protocols for using PCDHA8 antibodies in Western blotting?

For Western blotting applications using PCDHA8 antibodies, the following methodological approach is recommended:

Recommended Dilutions:

• Starting dilution: 1:1000 for most rabbit polyclonal anti-PCDHA8 antibodies

Protocol Outline:

• Sample Preparation:

  • Harvest cells or tissue and lyse in RIPA buffer containing protease inhibitors

  • Determine protein concentration using Bradford or BCA assay

  • Prepare samples (20-50μg total protein) in reducing sample buffer

• Gel Electrophoresis:

  • Separate proteins on 8-10% SDS-PAGE gel (appropriate for detecting the 160 kDa PCDHA8 protein)

  • Include molecular weight markers

• Transfer:

  • Transfer proteins to PVDF or nitrocellulose membrane at 100V for 60-90 minutes

• Blocking:

  • Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary Antibody Incubation:

  • Dilute PCDHA8 antibody 1:1000 in blocking solution

  • Incubate overnight at 4°C with gentle agitation

• Washing:

  • Wash membrane 3×10 minutes with TBST

• Secondary Antibody:

  • Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000-1:10000)

  • Incubate for 1 hour at room temperature

• Detection:

  • Develop using ECL substrate and image using film or digital imager

  • Expected molecular weight: approximately 160 kDa for full-length PCDHA8

When validating a new antibody, including positive and negative controls is essential. MCF-7 cells have been documented to express PCDHA8 and can serve as a positive control .

What are the optimal protocols for immunofluorescence detection of PCDHA8?

For immunofluorescence detection of PCDHA8, the following protocol is recommended based on published research methodologies:

Recommended Dilutions:

• Starting dilution: 1:10 to 1:50 for most anti-PCDHA8 antibodies in IF applications

Protocol for Cells:

• Cell Preparation:

  • Culture cells on coverslips to 50-70% confluence

  • Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

• Blocking:

  • Block with 1-5% BSA in PBS for 30-60 minutes at room temperature

• Primary Antibody:

  • Dilute PCDHA8 antibody (e.g., 10 μg/mL for monoclonal antibody MAB5434)

  • Incubate for 2-3 hours at room temperature or overnight at 4°C in a humidified chamber

• Washing:

  • Wash 3×5 minutes with PBS

• Secondary Antibody:

  • Incubate with fluorophore-conjugated secondary antibody (e.g., NorthernLights 557-conjugated Anti-Mouse/Rabbit IgG)

  • Incubate for 1 hour at room temperature in the dark

• Nuclear Counterstain:

  • Counterstain with DAPI (1 μg/mL) for 5 minutes

• Mounting:

  • Mount coverslips with anti-fade mounting medium

• Imaging:

  • Image using fluorescence microscopy

Expected Localization: PCDHA8 typically shows a pattern of cell surface and cytoplasmic staining . In neuronal cells, particular attention should be paid to synaptic membrane localization, while in epithelial cells, cell-cell junctions should be examined closely.

How can PCDHA8 antibodies be validated for specificity in experimental applications?

Validating PCDHA8 antibodies for specificity is crucial for ensuring reliable experimental results. A comprehensive validation strategy includes:

• Western Blot Analysis:

  • Confirm the antibody detects a band of appropriate molecular weight (approximately 160 kDa for full-length PCDHA8)

  • Check for absence of non-specific bands

  • Include positive control samples known to express PCDHA8 (e.g., MCF-7 cells)

• Peptide Competition Assay:

  • Pre-incubate the antibody with the immunizing peptide or recombinant protein (e.g., NBP1-86195PEP)

  • Perform parallel experiments with blocked and unblocked antibody

  • Specific signals should be significantly reduced in the presence of the blocking peptide

• Knockout/Knockdown Controls:

  • Test the antibody on samples with genetic knockout or siRNA knockdown of PCDHA8

  • Signal should be absent or significantly reduced in knockout/knockdown samples

• Cross-Reactivity Testing:

  • Test the antibody against related protocadherins to assess cross-reactivity

  • Particularly important given the high homology between different protocadherin family members

• Multiple Antibody Comparison:

  • Compare results using antibodies targeting different epitopes of PCDHA8

  • Consistent staining patterns strengthen confidence in specificity

• Immunoprecipitation Followed by Mass Spectrometry:

  • For definitive validation, perform IP with the antibody followed by mass spectrometry

  • Confirm the presence of PCDHA8 peptides in the immunoprecipitated material

A thorough validation should include at least three of these approaches to ensure antibody specificity before proceeding with complex experiments or attempting to publish results.

How can PCDHA8 antibodies be used to investigate splice variants and post-translational modifications?

Investigating PCDHA8 splice variants and post-translational modifications requires sophisticated experimental approaches utilizing specific antibodies:

For Splice Variant Detection:

• Epitope-Specific Antibodies:

  • Choose antibodies targeting regions present in specific splice variants

  • For example, antibodies recognizing regions within aa 134-607 or 781-877 would not detect variants with deletions in these regions

• Western Blot Protocol for Splice Variant Discrimination:

  • Use higher resolution SDS-PAGE (6-8% gels with longer run times)

  • Include positive controls expressing known splice variants

  • Expected molecular weights:

    • Full-length PCDHA8: ~160 kDa

    • Variant with aa 134-607 deletion: ~90 kDa

    • Variant with aa 781-877 deletion: ~150 kDa

• RT-PCR Validation:

  • Design primers flanking potential splice regions

  • Confirm protein findings with transcript evidence

For Post-Translational Modification (PTM) Analysis:

• Phosphorylation Studies:

  • Immunoprecipitate PCDHA8 using validated antibodies

  • Probe with anti-phospho-serine/threonine/tyrosine antibodies

  • Alternatively, use phosphatase treatment prior to Western blotting to identify mobility shifts

• Glycosylation Analysis:

  • Treat samples with glycosidases (PNGase F for N-linked; O-glycosidase for O-linked glycans)

  • Compare mobility shifts before and after treatment by Western blot

• Proteolytic Processing:

  • Compare antibodies targeting different domains to detect potential cleaved fragments

  • For example, compare N-terminal vs. C-terminal targeting antibodies to detect ectodomain shedding

• Mass Spectrometry Approaches:

  • Immunoprecipitate PCDHA8 using validated antibodies

  • Perform LC-MS/MS analysis to identify PTMs

  • Target the extracellular cadherin domains (aa 30-723) which may contain functional modifications

These advanced approaches allow researchers to move beyond simple protein detection to understand the complex regulation of PCDHA8 through alternative splicing and post-translational modifications, providing insights into its diverse functions in different cellular contexts.

What experimental approaches can be used to study PCDHA8's role in neuronal cell-cell connections?

Studying PCDHA8's role in neuronal cell-cell connections requires integrating antibody-based techniques with functional neuronal assays:

1. High-Resolution Localization Studies:

• Super-resolution microscopy with PCDHA8 antibodies to visualize precise synaptic localization

• Immunogold electron microscopy to determine exact subcellular localization at synapses

• Protocol elements:

  • Use monoclonal antibodies for higher specificity

  • Counter-label with synaptic markers (e.g., PSD-95, synaptophysin)

  • Quantify co-localization using appropriate statistical methods

2. Functional Manipulation Combined with Antibody Labeling:

• Electrophysiological recording of LTP with simultaneous or subsequent immunofluorescence

• Experimental design:

  • Perform patch-clamp recordings in hippocampal slices

  • Induce LTP using standard protocols

  • Fix tissue and perform immunofluorescence for PCDHA8

  • Correlate PCDHA8 expression/localization with electrophysiological parameters

3. Calcium Imaging with PCDHA8 Detection:

• Protocol:

  • Load neurons with calcium indicators (Fluo-4 or GCaMP)

  • Image calcium dynamics during synaptic activity

  • Fix cells and perform immunofluorescence for PCDHA8

  • Analyze correlation between PCDHA8 localization and calcium signaling patterns

4. Blocking Function with Function-Blocking Antibodies:

• Generate or obtain antibodies targeting the extracellular cadherin domains (aa 30-723)

• Apply to neuronal cultures to disrupt homophilic interactions

• Assess effects on:

  • Synapse formation (using synapse quantification)

  • Synaptic transmission (using electrophysiology)

  • Neuronal network activity (using multi-electrode arrays)

5. Proximity Ligation Assay (PLA) to Detect Protein Interactions:

• Protocol:

  • Use PCDHA8 antibody in combination with antibodies against potential binding partners

  • Perform PLA according to manufacturer's instructions

  • Quantify interaction signals at synaptic sites

  • Potential interacting proteins to investigate: other protocadherins, classical cadherins, synaptic scaffolding proteins

These methodological approaches, when properly controlled and quantified, can provide significant insights into PCDHA8's specific roles in establishing and maintaining neuronal connections, contributing to our understanding of synaptic specificity and plasticity.

How can antibodies be used to investigate PCDHA8's involvement in cell adhesion and tissue architecture?

Investigating PCDHA8's involvement in cell adhesion and tissue architecture requires specialized experimental approaches utilizing well-validated antibodies:

1. 3D Cellular Aggregation Assays:

• Protocol:

  • Culture cells expressing PCDHA8 in hanging drops or ultra-low attachment plates

  • Process for immunofluorescence staining with anti-PCDHA8 antibodies

  • Use confocal microscopy to analyze PCDHA8 distribution at cell-cell contacts

  • Compare aggregation patterns between control and PCDHA8-depleted cells

  • Expected results: PCDHA8 should concentrate at homophilic cell-cell contacts, and aggregation may be impaired in PCDHA8-depleted cells

2. Tissue Architecture Analysis:

• Experimental approach:

  • Perform immunohistochemistry on tissue sections (particularly breast epithelium where PCDHA8 is expressed)

  • Start with 1:10-50 dilution for IHC-P applications

  • Counter-stain with epithelial markers (E-cadherin, cytokeratins)

  • Analyze co-localization at tissue junctions

  • Data collection: Quantify PCDHA8 expression patterns in relation to architectural features using image analysis software

3. Calcium-Dependence of PCDHA8 Adhesion:

• Protocol:

  • Perform adhesion assays with cells expressing PCDHA8

  • Manipulate calcium levels (0-2mM Ca²⁺)

  • Fix and stain with anti-PCDHA8 antibodies

  • Analysis: Quantify adhesion strength and PCDHA8 localization as a function of calcium concentration

4. Cell Migration and Wound Healing:

• Experimental design:

  • Perform scratch wound assays on epithelial monolayers

  • Monitor wound closure by time-lapse microscopy

  • Fix at various time points and stain for PCDHA8

  • Expected patterns: PCDHA8 may redistribute during the migration and re-establishment of cell-cell contacts

5. Interaction with Cytoskeleton:

• Protocol:

  • Co-immunoprecipitate PCDHA8 using validated antibodies

  • Probe for cytoskeletal components (actin, microtubules)

  • Alternatively, perform dual immunofluorescence for PCDHA8 and cytoskeletal elements

  • Data interpretation: Assess whether PCDHA8 colocalizes with or affects cytoskeletal organization at adhesion sites

These methodologies allow researchers to move beyond simple localization studies to understand the functional roles of PCDHA8 in establishing and maintaining tissue architecture, providing insights into both normal development and potential pathological changes in diseases such as cancer where cell adhesion is disrupted.

What are common issues when using PCDHA8 antibodies and how can they be resolved?

Researchers often encounter several challenges when working with PCDHA8 antibodies. Here are common issues and their methodological solutions:

1. High Background in Immunostaining:

2. Weak or No Signal in Western Blot:

3. Cross-Reactivity with Other Protocadherins:

4. Inconsistent Results Between Experiments:

These troubleshooting approaches should be systematically documented and incorporated into experimental protocols to ensure reproducible results with PCDHA8 antibodies.

How should researchers interpret contradictory results from different PCDHA8 antibodies?

When faced with contradictory results from different PCDHA8 antibodies, researchers should employ a systematic approach to interpretation and validation:

1. Epitope Mapping Analysis:

First, determine the exact epitopes recognized by each antibody. The following table summarizes considerations:

2. Methodological Reconciliation Framework:

When different antibodies yield contradictory results, implement this decision tree:

• Compare validation data for each antibody:

  • How extensively was each antibody validated?

  • Are there peptide competition results available?

  • Are knockout/knockdown controls available?

• Evaluate technical variables:

  • Different fixation methods may preserve different epitopes

  • Antigen retrieval methods can influence epitope accessibility

  • Sample preparation may affect protein confirmation

• Consider biological explanations:

  • Different splice variants may be expressed in different tissues

  • Post-translational modifications may mask certain epitopes

  • Protein interactions may hide epitopes in a context-dependent manner

3. Synthesis Approach for Contradictory Data:

When contradictions persist, implement this validation strategy:

• Orthogonal techniques:

  • Validate protein expression using mRNA analysis (RT-PCR, RNA-Seq)

  • Use epitope-tagged PCDHA8 constructs for overexpression studies

  • Employ mass spectrometry to definitively identify protein forms

• Functional validation:

  • Use CRISPR/Cas9 to knockout PCDHA8 and assess antibody specificity

  • Perform rescue experiments with defined PCDHA8 constructs

• Cautious interpretation:

  • Report all findings with appropriate caveats

  • Present data from multiple antibodies alongside controls

  • Be transparent about limitations in publications

By approaching contradictory results methodically, researchers can extract valuable information about the complex biology of PCDHA8, including its processing, modifications, and interactions, rather than simply dismissing contradictory results as technical failures.

What considerations are important when analyzing PCDHA8 expression across different tissue and cell types?

Analyzing PCDHA8 expression across different tissue and cell types requires careful methodological consideration and data interpretation:

1. Tissue-Specific Expression Pattern Considerations:

2. Technical Considerations for Cross-Tissue Comparison:

• Fixation optimization:

  • Different tissues may require different fixation protocols

  • Test multiple fixatives (4% PFA, methanol, acetone)

  • Optimize antigen retrieval methods for each tissue type

• Antibody validation per tissue type:

  • Validate antibody specificity in each new tissue context

  • Include tissue-specific negative controls (e.g., PCDHA8-negative tissue regions)

  • Consider using multiple antibodies targeting different epitopes

• Quantification strategies:

  • Develop tissue-specific quantification parameters

  • Account for autofluorescence differences between tissues

  • Use internal controls for normalization

3. Biological Interpretation Framework:

• Expression level differences:

  • High expression in neurons aligns with role in synaptic specificity

  • Expression in epithelial cells relates to architectural functions

  • Quantify relative expression across tissues using calibrated Western blotting

• Subcellular localization variations:

  • Membrane localization suggests active adhesion function

  • Cytoplasmic accumulation may indicate internalization or processing

  • Nuclear localization could suggest non-canonical signaling roles

• Co-expression analysis:

  • Examine co-expression with other protocadherin family members

  • Assess relationship with classical cadherins and catenins

  • Use multiplexed immunofluorescence for co-localization studies

• Splice variant distribution:

  • Different tissues may express distinct splice variants

  • Design experiments to discriminate between variants

  • Consider RNA analysis to complement protein studies

By systematically addressing these considerations, researchers can generate comprehensive and accurate profiles of PCDHA8 expression across different tissues, leading to deeper insights into its diverse biological functions in different cellular contexts.

How can PCDHA8 antibodies be utilized in studying neurodevelopmental disorders?

PCDHA8 antibodies offer powerful tools for investigating potential connections between protocadherins and neurodevelopmental disorders, with several methodological approaches showing promise:

1. Comparative Expression Analysis in Normal vs. Disorder Models:

Utilizing immunohistochemistry with anti-PCDHA8 antibodies (starting at 1:10-50 dilution) , researchers can:

• Compare PCDHA8 expression patterns in post-mortem brain tissues from individuals with neurodevelopmental disorders versus controls

• Analyze expression in animal models of neurodevelopmental disorders

• Investigate specific brain regions implicated in disorders (prefrontal cortex, hippocampus, cerebellum)

2. Single-Cell Analysis in Development:

• Combine anti-PCDHA8 immunostaining with markers of neuronal subtypes

• Map PCDHA8 expression during critical developmental windows

• Compare developmental trajectory in normal vs. disorder models

• Analyze potential differential expression in excitatory vs. inhibitory neurons

3. Synaptic Connectivity Assessment:

4. Genetic Variant Characterization:

• Generate antibodies specific to common PCDHA8 variants associated with disorders

• Express variant forms in cellular models and examine trafficking/localization

• Assess functional consequences on adhesion and signaling properties

5. Therapeutic Targeting Approaches:

• Use antibodies to screen for compounds that modulate PCDHA8 function

• Develop blocking or activating antibodies for functional studies

• Investigate whether restoring normal PCDHA8 levels/function can rescue phenotypes

This research direction offers significant potential for understanding how cell adhesion molecules contribute to the synaptic and circuit-level abnormalities observed in neurodevelopmental disorders, potentially leading to novel diagnostic approaches or therapeutic targets.

What are the most promising techniques for studying PCDHA8 interactions with other protocadherins?

Studying PCDHA8 interactions with other protocadherins requires sophisticated methodological approaches that can detect specific molecular interactions while maintaining physiological relevance:

1. Advanced Co-Immunoprecipitation Approaches:

Protocol elements:

• Use anti-PCDHA8 antibodies for pulldown experiments

• Follow with mass spectrometry to identify interacting protocadherins

• Validate interactions using reverse co-IP with antibodies against identified partners

2. Microscopy-Based Interaction Analysis:

Proximity Ligation Assay (PLA) protocol:

• Fix cells/tissues using 4% paraformaldehyde

• Incubate with primary antibodies against PCDHA8 and potential interacting protocadherins

• Apply PLA probes and perform ligation and amplification

• Analyze PLA signals that appear as fluorescent dots where proteins are in close proximity (<40nm)

FRET/FLIM analysis:

• Express fluorescently-tagged PCDHA8 and potential partners

• Measure energy transfer as indicator of protein-protein interaction

• Complement with antibody staining to verify physiological relevance

3. Biochemical Characterization of Interactions:

Surface Plasmon Resonance (SPR) protocol:

• Immobilize purified PCDHA8 extracellular domain on sensor chip

• Flow solutions containing extracellular domains of other protocadherins

• Measure binding kinetics and affinities

• Use specific antibodies to confirm protein identity/function

Analytical Ultracentrifugation:

• Mix purified protocadherin ectodomains

• Analyze complex formation under various conditions (Ca²⁺ concentrations)

• Use domain-specific antibodies to verify identities in complexes

4. Functional Validation of Interactions:

Cell aggregation assays:

• Express different combinations of protocadherins in separate cell populations

• Mix cells and assess formation of homophilic vs. heterophilic aggregates

• Use antibodies against specific protocadherins to disrupt interactions

• Quantify aggregation patterns and correlation with protocadherin expression

These methodological approaches, when combined, provide a comprehensive framework for understanding the complex interactions between PCDHA8 and other protocadherins, leading to insights into how these molecules contribute to the "protocadherin code" that may underlie neuronal connectivity specificity.

What are the most significant open questions regarding PCDHA8 function that antibody-based research could address?

Several critical questions about PCDHA8 function remain unanswered, and antibody-based approaches offer promising avenues for addressing these knowledge gaps:

• Synaptic Specificity Mechanisms: How does PCDHA8 contribute to the molecular code for synaptic specificity? Utilizing highly specific PCDHA8 antibodies in combination with high-resolution imaging techniques could help map the precise distribution of PCDHA8 at synapses in relation to other protocadherins, potentially revealing patterns that correlate with specific circuit connections .

• Signaling Pathway Integration: What downstream signaling pathways are activated by PCDHA8-mediated adhesion? Phospho-specific antibodies targeting potential phosphorylation sites on PCDHA8, combined with pull-down approaches to identify interacting signaling molecules, could elucidate how adhesion events are translated into cellular responses.

• Regulatory Mechanisms: How is PCDHA8 expression spatiotemporally regulated during development and in response to activity? Antibody-based chromatin immunoprecipitation (ChIP) approaches targeting transcription factors that potentially regulate PCDHA8 expression could reveal regulatory mechanisms.

• Pathological Relevance: What role does PCDHA8 dysfunction play in neurodevelopmental disorders or cancer progression? Comparative studies using validated antibodies to analyze expression patterns in normal versus diseased tissues could identify correlations with pathological states.

• Therapeutic Potential: Could modulation of PCDHA8 function serve as a therapeutic approach? Development of function-blocking or function-enhancing antibodies could provide tools to test this hypothesis in model systems.

Addressing these questions will require continued development and validation of high-quality, specific antibodies against PCDHA8 and related proteins, as well as integration of antibody-based approaches with complementary molecular, cellular, and systems-level techniques.

How might emerging antibody technologies enhance PCDHA8 research in the next decade?

Emerging antibody technologies promise to revolutionize PCDHA8 research in the coming decade, offering unprecedented tools for detection, functional manipulation, and therapeutic development:

1. Next-Generation Antibody Development Technologies:

2. Advanced Imaging Applications:

• Super-resolution microscopy with quantum dot-conjugated antibodies:

  • Track single PCDHA8 molecules in living neurons

  • Map nanoscale distribution at synapses with 5-10nm resolution

  • Analyze dynamic changes during synapse formation and plasticity

• Expansion microscopy with multi-color antibody labeling:

  • Physically expand samples to achieve super-resolution with standard microscopes

  • Map complex protocadherin codes at individual synapses

  • Correlate with functional properties

3. Functional Manipulation Tools:

• Optogenetic antibody systems:

  • Develop photoswitchable antibodies that can block PCDHA8 function upon light stimulation

  • Enable temporally precise, spatially restricted functional manipulation

  • Combine with electrophysiology to directly test functional hypotheses

• PROTAC (Proteolysis Targeting Chimera) antibody conjugates:

  • Create antibody-based PROTACs targeting PCDHA8 for controlled degradation

  • Enable rapid, specific protein depletion without genetic manipulation

  • Study acute effects of PCDHA8 loss in mature circuits

4. Single-Cell Multi-Omic Integration:

• Antibody-based spatial transcriptomics:

  • Combine PCDHA8 antibody staining with in situ RNA sequencing

  • Correlate protein expression with transcriptional state in individual cells

  • Map cell type-specific expression patterns at unprecedented resolution

• Mass cytometry with PCDHA8 antibodies:

  • Simultaneously measure dozens of protein markers alongside PCDHA8

  • Identify cell populations with distinct protocadherin expression profiles

  • Correlate with functional states