PCDH1 Antibody

What is PCDH1 and what cellular functions does it serve?

Protocadherin 1 (PCDH1), also known as cadherin-like protein 1 or PC42, is a transmembrane protein that mediates calcium-dependent cell-cell adhesion. It belongs to the delta1-protocadherins group, which includes protocadherin-1, -7, -9, and -11(X/Y). PCDH1 features seven extracellular cadherin-repeats, a transmembrane domain, and a cytoplasmic domain . Its expression is regulated both temporally and spatially, playing essential roles in early development and mature tissues. PCDH1 is primarily localized at cell-cell and cell-matrix boundaries, with particularly high expression in brain and neuro-glial cells, suggesting its importance in neuronal development and synapse formation . Additionally, PCDH1 has been identified as a susceptibility gene for Bronchial Hyperresponsiveness in humans and is expressed on human pluripotent stem cells .

What types of PCDH1 antibodies are available for research applications?

Several types of PCDH1 antibodies are available for research applications, including:

• Monoclonal antibodies such as CSTEM31, which reacts with human Protocadherin-1 and is suitable for flow cytometry and immunocytochemistry .

• Mouse monoclonal antibodies like B-11 (IgG1 kappa light chain), which detect PCDH1 protein from mouse, rat, and human sources through western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) .

• Various conjugated forms including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates for specialized applications .

• Polyclonal antibodies that have been used in combination with monoclonal antibodies to validate PCDH1 expression patterns in different tissues .

For flow cytometry applications, pre-diluted antibodies like CSTEM31 can be used at approximately 5 μL (0.5 μg) per test, where a test is defined as the amount of antibody needed to stain a cell sample in a final volume of 100 μL .

What are the expression patterns of PCDH1 across different tissues?

PCDH1 exhibits distinct expression patterns across tissues and developmental stages. In mice, PCDH1 is expressed in a wide array of tissues and organs, including:

• Central nervous system: brain and spinal cord

• Peripheral nervous system: peripheral ganglia

• Sensory organs: inner ear

• Epidermal structures: hair follicles

• Excretory system: kidney

• Reproductive system: reproductive organs, prostate, and seminal gland

In humans, PCDH1 is highly expressed in brain and neuroglial cells, indicating its critical role in neuronal development and synapse formation . PCDH1 expression has also been detected in airway epithelial cells from asthma patients, consistent with its role as a susceptibility gene for Bronchial Hyperresponsiveness . Additionally, PCDH1 is expressed on human pluripotent stem cells, suggesting its importance in stem cell biology and developmental processes . Recent research has also identified upregulated PCDH1 expression in pancreatic ductal adenocarcinoma (PDAC) tissues, with expression levels correlating with tumor invasion depth and lymph node metastasis .

What are the optimal protocols for PCDH1 detection using immunocytochemistry?

For immunocytochemical detection of PCDH1, the following methodological approach is recommended:

• Fixation and permeabilization: For optimal results with human induced pluripotent stem cells (iPSCs), use formaldehyde fixation followed by permeabilization. This preparation method preserves PCDH1's native conformation while allowing antibody access to cellular targets .

• Antibody concentration: The CSTEM31 monoclonal antibody can be used at concentrations ≤5 μg/mL for immunocytochemistry applications .

• Detection system: Use an appropriate secondary antibody or visualization system compatible with your primary antibody. For fluorescent detection, select secondary antibodies with minimal spectral overlap if performing multiplex staining.

• Controls: Include both positive controls (cells known to express PCDH1, such as human iPSCs) and negative controls (omission of primary antibody or use of isotype control) to validate staining specificity.

• Counterstaining: Use nuclear counterstains (e.g., DAPI) to facilitate visualization of cell morphology and antibody localization.

• Imaging: Capture images using confocal or fluorescence microscopy with appropriate filter sets for your detection system .

How should researchers optimize PCDH1 antibody use in flow cytometry applications?

For effective flow cytometric analysis of PCDH1 expression, researchers should follow these methodological guidelines:

• Antibody selection: For flow cytometry, fluorochrome-conjugated antibodies like CSTEM31-PE are recommended. These pre-conjugated antibodies eliminate the need for secondary antibody staining, reducing background and non-specific binding .

• Titration: Determine the optimal antibody concentration through titration experiments. The recommended starting concentration for CSTEM31 is 5 μL (0.5 μg) per test, where a test is defined as the amount needed to stain a cell sample in 100 μL final volume .

• Cell number: While cell numbers can range from 10^5 to 10^8 cells/test, empirical determination is recommended for each system. Higher cell concentrations may require increased antibody amounts .

• Controls: Include:

  • Unstained cells to establish autofluorescence baseline

  • Isotype controls (matched to primary antibody isotype) to assess non-specific binding

  • FMO (fluorescence minus one) controls for multicolor panels

  • Positive controls using cell types known to express PCDH1 (e.g., human iPSCs)

• Instrument settings: Optimize excitation and emission settings based on your fluorochrome. For PE-conjugated PCDH1 antibodies, use:

  • Excitation: 488-561 nm

  • Emission: 578 nm

  • Compatible lasers: Blue, Green, or Yellow-Green

• Compensation: For multicolor experiments, proper compensation is essential to correct for spectral overlap between fluorochromes.

What approaches should be used to validate PCDH1 antibody specificity?

Validating PCDH1 antibody specificity is crucial for generating reliable research data. Recommended validation approaches include:

• Multiple antibody comparison: Use both polyclonal and monoclonal antibodies targeting different PCDH1 epitopes and compare staining patterns. Concordant results increase confidence in specificity .

• Genetic validation:

  • Use PCDH1 knockout or knockdown models as negative controls

  • Perform rescue experiments by reintroducing PCDH1 expression in knockout systems

  • Compare staining patterns in wild-type versus genetically modified samples

• Peptide competition assays: Pre-incubate the antibody with purified PCDH1 protein or peptide before application to samples. Specific binding should be blocked by competition .

• Domain-specific antibodies: Use antibodies targeting specific domains (e.g., EC1-specific antibody 3305) to validate domain-specific interactions. This approach is particularly useful for functional studies where specific domains are implicated in biological processes .

• Western blot validation: Confirm antibody specificity by western blot, verifying that bands appear at the expected molecular weights for PCDH1 isoforms (which exist in short and long forms due to alternative splicing) .

• Cross-reactivity testing: Test antibodies against related protocadherins to ensure specificity within the protocadherin family.

How are PCDH1 antibodies used in bronchial hyperresponsiveness research?

PCDH1 has been identified as a novel susceptibility gene for bronchial hyperresponsiveness, making PCDH1 antibodies valuable tools in respiratory disease research. Methodological approaches include:

• Expression analysis: PCDH1 antibodies can be used to compare protein expression levels between healthy controls and patients with respiratory disorders. Both western blotting and immunohistochemistry have been employed to analyze PCDH1 expression in lung tissues and airway epithelial cells from asthma patients .

• Cell-specific expression: Researchers can employ immunofluorescence with PCDH1 antibodies to determine which specific cell types in the airway express PCDH1, helping to elucidate the mechanism by which PCDH1 contributes to bronchial hyperresponsiveness.

• Co-localization studies: PCDH1 antibodies can be used in conjunction with markers for specific cell types or subcellular structures to understand PCDH1's localization and potential interaction partners in airway cells.

• Tissue-specific expression: PCR and immunohistochemistry with PCDH1-specific antibodies have been used to investigate PCDH1 expression in epithelial cell lines (16HBE), blood cells, brain, lung fibroblasts, and cultured airway epithelial cells from asthma patients .

• Functional blocking studies: PCDH1-specific antibodies could potentially be used to block PCDH1 function in cell culture or animal models to assess its role in airway hyperreactivity and inflammation.

What role does PCDH1 play in cancer progression and how can antibodies help study this function?

PCDH1 has been implicated in cancer progression, particularly in pancreatic ductal adenocarcinoma (PDAC). PCDH1 antibodies can facilitate cancer research through the following methodological approaches:

How can researchers utilize PCDH1 antibodies to study viral infection mechanisms?

PCDH1 has been identified as essential for cell entry by New World hantaviruses, making PCDH1 antibodies valuable tools for studying viral infection mechanisms:

• Viral entry blocking assays: EC1-specific antibodies (such as antibody 3305) can be used to block New World hantavirus entry into human cells. This approach has demonstrated that preincubation of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Pulmonary Microvascular Endothelial Cells (HPMECs) with EC1-specific antibody specifically inhibits Gn/Gc-dependent entry by New World viruses .

• Domain mapping: Using domain-specific antibodies targeting different PCDH1 extracellular cadherin-repeat domains helps determine which domains are critical for viral entry. Research has shown that the first extracellular cadherin-repeat domain (EC1) is particularly important for New World hantavirus entry .

• Virus-receptor interaction studies: PCDH1 antibodies can be used in competition assays to study the interaction between viral glycoproteins (Gn/Gc) and PCDH1. When cells expressing Andes virus (ANDV) Gn/Gc were incubated with soluble EC1-2 fragments, only ANDV Gn/Gc-expressing cells could be decorated with sEC1-2, and this capture was sensitive to EC1-specific antibody 3305 .

• ELISA-based viral capture assays: Immobilized soluble PCDH1 EC1-2 fragments can selectively capture virus particles bearing Gn/Gc from New World hantaviruses (ANDV and SNV) in a manner that is sensitive to EC1-specific antibody 3305 .

• Virus tropism studies: PCDH1 antibodies can help identify tissues and cell types expressing PCDH1, potentially explaining the tropism of New World hantaviruses.

What are common technical challenges when using PCDH1 antibodies and how can they be addressed?

When working with PCDH1 antibodies, researchers may encounter several technical challenges. Here are methodological solutions to address them:

• Variable detection sensitivity:

  • Optimize antibody concentration through titration experiments

  • Try different antibody clones if one shows poor sensitivity

  • For flow cytometry, use brighter fluorochromes (PE or APC) rather than dimmer ones (FITC)

  • Consider signal amplification methods for low-expression samples

• High background or non-specific binding:

  • Include appropriate blocking steps (serum from the same species as secondary antibody)

  • Use more stringent washing conditions

  • Titrate primary and secondary antibodies to optimal concentrations

  • For flow cytometry, include viability dyes to exclude dead cells which can bind antibodies non-specifically

• Isoform-specific detection:

  • Select antibodies that can distinguish between short and long PCDH1 isoforms if isoform specificity is important

  • Use western blotting to confirm which isoforms are being detected

  • Consider using isoform-specific primers for qPCR validation of protein findings

• Fixation artifacts:

  • Test multiple fixation methods as PCDH1 epitopes may be sensitive to certain fixatives

  • For immunocytochemistry on iPSCs, formaldehyde fixation followed by permeabilization has been validated

  • Consider native (non-fixed) conditions for flow cytometry if membrane integrity is not compromised

• Species cross-reactivity:

  • Validate antibodies for your specific species of interest

  • The B-11 antibody has been validated for mouse, rat, and human PCDH1 , while some antibodies may be human-specific

How should researchers determine the optimal PCDH1 antibody for their specific application?

Selecting the optimal PCDH1 antibody requires careful consideration of several methodological factors:

• Application compatibility:

  • For flow cytometry: Pre-conjugated antibodies (like CSTEM31-PE) are recommended

  • For western blotting: Antibodies validated for denatured proteins (like B-11)

  • For immunoprecipitation: Antibodies available in agarose-conjugated format

  • For immunofluorescence: Antibodies validated for fixed tissues/cells

• Species reactivity:

  • Ensure the antibody recognizes PCDH1 in your species of interest

  • B-11 antibody detects mouse, rat, and human PCDH1

  • CSTEM31 is validated for human PCDH1

• Domain specificity:

  • For functional blocking studies: EC1-specific antibodies (like antibody 3305) are effective for blocking hantavirus entry

  • For general detection: Antibodies targeting conserved regions may be preferable

• Clonality considerations:

  • Monoclonal antibodies: Higher specificity, less batch-to-batch variation

  • Polyclonal antibodies: Potentially higher sensitivity by recognizing multiple epitopes

• Validation evidence:

  • Review published literature using the antibody for your application

  • Check validation data from manufacturers (western blots, flow cytometry profiles)

  • Consider antibodies validated by knockout/knockdown controls

• Format requirements:

  • Consider conjugation needs (fluorochromes, enzymes, biotin)

  • For multicolor flow cytometry, select fluorochromes compatible with your instrument and panel

What controls should be included when using PCDH1 antibodies for research?

Proper controls are essential for generating reliable data with PCDH1 antibodies. The following methodological controls should be considered:

• Positive controls:

  • Cell lines known to express PCDH1 (e.g., human iPSCs, brain-derived cell lines)

  • Tissues with documented PCDH1 expression (brain, spinal cord, lung epithelial cells)

  • Recombinant PCDH1 protein (for western blotting)

• Negative controls:

  • Isotype controls matched to the primary antibody class and subclass

  • PCDH1 knockout or knockdown cells/tissues (genetically validated)

  • Primary antibody omission controls

• Specificity controls:

  • Peptide competition/blocking experiments

  • Use of multiple antibodies targeting different PCDH1 epitopes

  • Western blot validation of antibody specificity before use in other applications

• Application-specific controls:

  • For flow cytometry: Fluorescence Minus One (FMO) controls, compensation controls

  • For immunofluorescence: Autofluorescence controls

  • For western blotting: Loading controls, molecular weight markers

• Functional validation:

  • For antibodies used in blocking experiments (like EC1-specific antibody 3305), include functional readouts before and after antibody treatment

  • For antibodies used in diagnostic applications, include samples with known clinical outcomes

How can PCDH1 antibodies contribute to stem cell research?

PCDH1 has been identified on human pluripotent stem cells, offering several methodological applications for stem cell research:

• Stem cell characterization:

  • PCDH1 antibodies like CSTEM31 can be used to characterize human induced pluripotent stem cells (iPSCs) through flow cytometry and immunocytochemistry

  • Include PCDH1 in multiparameter flow cytometry panels alongside established pluripotency markers

• Developmental studies:

  • Track PCDH1 expression changes during differentiation from pluripotent to specialized cell types

  • Use time-course immunofluorescence or flow cytometry with PCDH1 antibodies to monitor temporal expression patterns

• Cell sorting applications:

  • Fluorochrome-conjugated PCDH1 antibodies can potentially be used for fluorescence-activated cell sorting (FACS) to isolate specific stem cell populations

  • Combine with other markers for refined subset identification

• Functional investigations:

  • Use blocking PCDH1 antibodies to study its role in stem cell adhesion, migration, and differentiation

  • Apply in stem cell culture to assess effects on colony formation and maintenance

• Regenerative medicine applications:

  • Evaluate PCDH1 expression in therapeutic stem cell products as a quality control measure

  • Investigate PCDH1's potential role in tissue engineering applications

What is the potential for PCDH1 antibodies in developing therapeutic approaches?

While PCDH1 antibodies are currently research tools, their therapeutic potential is emerging across several areas:

• Antiviral therapy development:

  • EC1-specific antibodies like antibody 3305 can block New World hantavirus entry into human cells

  • These findings suggest potential for therapeutic antibodies that target PCDH1 EC1 domain to prevent or treat hantavirus infections

  • Research models could employ these antibodies to validate therapeutic approaches

• Cancer therapeutics:

  • High PCDH1 expression correlates with poor prognosis in pancreatic ductal adenocarcinoma (PDAC)

  • Antibodies targeting PCDH1 could potentially inhibit tumor progression and metastasis

  • Researchers can use existing antibodies to validate PCDH1 as a therapeutic target in preclinical models

• Diagnostic applications:

  • PCDH1 antibodies may help identify patients with high PCDH1 expression who might benefit from targeted therapies

  • Immunohistochemistry with PCDH1 antibodies could aid in cancer prognosis and treatment planning

• Respiratory disease applications:

  • Given PCDH1's role as a susceptibility gene for bronchial hyperresponsiveness , antibodies blocking specific PCDH1 functions might have therapeutic potential

  • Research models using PCDH1 antibodies can help elucidate mechanisms for potential therapeutic development

• Antibody engineering:

  • Current research antibodies provide structural and functional insights that could inform the development of therapeutic antibodies with optimized properties

  • Domain-specific antibodies offer templates for developing more targeted therapeutic approaches