PCDHA10 Antibody, HRP conjugated

What is PCDHA10 and what cellular functions does it perform?

PCDHA10 is a member of the protocadherin alpha gene cluster, which consists of 15 cadherin superfamily genes related to mouse CNR genes. These neural cadherin-like cell adhesion proteins function as integral plasma membrane proteins that play critical roles in establishing and maintaining specific cell-cell connections in the brain . The protocadherin alpha gene cluster demonstrates an unusual genomic organization similar to B-cell and T-cell receptor gene clusters, with 13 highly similar and 2 more distantly related coding sequences .

Each gene in this cluster contains large, uninterrupted N-terminal exons encoding six cadherin ectodomains, while shared C-terminal exons encode the cytoplasmic domain . Alternative splicing has been observed in PCDHA10, though the complete characterization of all variants remains incomplete . While PCDHA10's specific functions are still being elucidated, research on related protocadherins suggests involvement in neuronal development, synaptic connectivity, and potentially tumor suppression pathways.

What applications are most suitable for PCDHA10 antibodies with HRP conjugation?

HRP-conjugated PCDHA10 antibodies are particularly valuable for applications requiring direct detection without secondary antibodies. Based on manufacturer specifications, these antibodies perform optimally in:

• Western blotting (recommended dilution 1:500-1:2000)

• Enzyme-linked immunosorbent assays (ELISA)

• Immunohistochemistry (IHC)

• Flow cytometry

Western blot analysis using PCDHA10 polyclonal antibody at 1:1000 dilution in U-87MG cell extracts has successfully detected PCDHA10 at the expected molecular weight of approximately 100kDa . The HRP conjugation provides direct enzymatic detection capability, eliminating the need for secondary antibody incubation steps and potentially reducing background signal in sensitive applications.

For immunofluorescence applications, a starting dilution of 1:50 with a working range of 1:50-1:500 has been reported for non-conjugated versions of the antibody, which may serve as a reference point for HRP-conjugated variants with appropriate modifications to detection protocols .

How should PCDHA10 HRP-conjugated antibodies be stored to maintain activity?

Proper storage is critical for maintaining antibody activity. For PCDHA10 antibodies:

• Store at -20°C

• Avoid freeze/thaw cycles that can degrade antibody quality and reduce sensitivity

• The antibody is typically provided in PBS buffer containing 0.01% thiomersal and 50% glycerol at pH 7.3, which helps maintain stability during storage

When working with the antibody, aliquoting into single-use volumes upon receipt is recommended to minimize freeze/thaw cycles. For short-term use (within one week), storage at 4°C is acceptable, but return to -20°C for long-term preservation of activity. Centrifuge vials before opening to ensure complete recovery of contents, particularly after shipping or extended storage .

What controls should be included when using PCDHA10 HRP-conjugated antibodies?

Proper experimental controls are essential for validating results with PCDHA10 antibodies:

• Positive control: Cell lines known to express PCDHA10, such as U-87MG cells, which have been documented to express detectable levels of PCDHA10 protein

• Negative control: One of the following:

  • Samples from knockout models lacking PCDHA10 expression

  • Cell lines with confirmed absence of PCDHA10 expression

  • Primary antibody omission control to assess non-specific binding of detection reagents

• Blocking peptide control: Competition studies can be performed using specific blocking peptides (such as sc-109747 P, 100 μg peptide in 0.5 ml PBS containing < 0.1% sodium azide and 0.2% BSA) to confirm antibody specificity

• siRNA knockdown validation: PCDHA10 siRNA (e.g., sc-106367) can be used to reduce PCDHA10 expression in appropriate cell lines, providing a control to verify antibody specificity in detecting the target protein

How can I optimize Western blot protocols for PCDHA10 detection using HRP-conjugated antibodies?

Western blot optimization for PCDHA10 detection requires attention to several methodological details:

• Sample preparation:

  • Use RIPA buffer with protease inhibitor cocktail

  • Load 20-50 μg of total protein per lane

  • Include phosphatase inhibitors if phosphorylation status is relevant

• Gel selection and transfer:

  • Use 8-10% SDS-PAGE gels for optimal separation around the 100kDa region where PCDHA10 is expected

  • Transfer to PVDF membranes at 100V for 60-90 minutes in cold transfer buffer with 10-20% methanol

• Blocking and antibody incubation:

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

  • For HRP-conjugated antibodies, use recommended dilutions (1:500-1:2000)

  • Incubate overnight at 4°C for primary antibody binding

  • Wash thoroughly (3-5 times, 5-10 minutes each) with TBST

• Detection optimization:

  • Use enhanced chemiluminescence (ECL) detection systems

  • Exposure times may need adjustment based on expression levels

  • For weak signals, consider substrate with extended signal duration

Expected molecular weight for PCDHA10 is approximately 100kDa as observed in U-87MG cell extracts . If multiple bands appear, validation with additional techniques such as immunoprecipitation or mass spectrometry may be necessary to confirm specificity.

What methodologies can be used to investigate PCDHA10's role in cell-cell interactions?

Investigating PCDHA10's role in cell-cell interactions requires multiple complementary approaches:

• Immunofluorescence co-localization studies:

  • Use PCDHA10 antibodies alongside markers for cell junctions or membranes

  • Starting dilution of 1:50 with range of 1:50-1:500 has been reported

  • Co-stain with antibodies against other adhesion molecules (e.g., PECAM-1) to examine co-localization at cell-cell junctions

• Functional adhesion assays:

  • Cell aggregation assays with cells expressing or lacking PCDHA10

  • Cell sorting assays to examine homophilic binding preference

  • Atomic force microscopy to measure adhesive forces between cells

• Loss-of-function approaches:

  • siRNA knockdown using validated reagents such as sc-106367 for mouse PCDHA10

  • CRISPR-Cas9 gene editing to create knockout cell lines

  • Monitor changes in cell-cell adhesion, morphology, and migratory behavior

• Co-immunoprecipitation assays:

  • Immunoprecipitate PCDHA10 to identify binding partners

  • Analyze interactions using techniques similar to those employed for other protocadherins like PCDH10

  • Use appropriate controls to confirm specificity of interactions

The methodology for siRNA knockdown validation has been described using mouse mammary gland cell lines, where siRNA treatment followed by downstream validation via qPCR and functional assays can effectively demonstrate PCDHA10's role in cellular processes .

How does PCDHA10 expression relate to human disease, particularly in the cardiovascular context?

Emerging research suggests important connections between PCDHA gene cluster variants and cardiovascular conditions:

• PCDHA deletion copy number variants (delCNVs) and cardiovascular development:

  • Two common deletion CNVs within the PCDHA gene cluster have been associated with left ventricular outflow tract obstruction (LVOTO)

  • Analysis of 1,218 individuals with LVOTO versus 463 control individuals yielded odds ratios of:

    • 1.47 (95% CI, 1.13–1.92; p = 4.2 × 10^-3) for LVOTO

    • 1.47 (95% CI, 1.10–1.97; p = 0.01) for bicuspid aortic valve (BAV)

    • 6.13 (95% CI, 2.75–13.7; p = 9.7 × 10^-6) for coarctation of aorta (CoA)

    • 1.49 (95% CI, 1.07–2.08; p = 0.019) for hypoplastic left heart syndrome (HLHS)

• Detection methodologies for PCDHA variants:

  • Multiplexed PCR assays have been developed to detect specific deletion variants:

    • 16.8 kb delCNV (nsv4655880) yields a 554 bp PCR product compared to 469 bp for wild-type

    • 13.6 kb delCNV (nsv4684081) yields a 2,500-3,000 bp PCR product compared to 1,440 bp for wild-type

  • These PCR genotyping protocols have been validated through comparison with whole exome sequencing data

• Functional studies in animal models:

  • Mouse models have shown that mutations in the Pcdha gene cluster can cause BAV and other LVOTO phenotypes

  • Immunostaining of mouse embryonic heart tissue using alpha-protocadherin antibodies (1:500 dilution) alongside PECAM-1 and alpha smooth muscle actin antibodies has been used to examine expression patterns during development

These findings suggest that PCDHA10 antibodies may have important applications in cardiovascular research beyond their traditional use in neuroscience, potentially serving as tools for examining protocadherin expression in developing and adult cardiac tissues.

What methodological considerations are important when using PCDHA10 antibodies for co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) studies with PCDHA10 require careful attention to methodology:

• Antibody selection and validation:

  • Confirm the antibody is suitable for immunoprecipitation applications

  • Validate using positive controls expressing known levels of PCDHA10

  • Consider using affinity-purified antibodies like the goat polyclonal PCDHA10 (S-12) antibody for optimal specificity

• Lysis buffer optimization:

  • Use mild lysis conditions to preserve protein-protein interactions

  • Typical buffer composition: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA with protease inhibitors

  • Avoid harsh detergents that may disrupt membrane protein interactions

• Protocol methodology:

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

  • Incubate with 2-5 μg of PCDHA10 antibody per 1 mg of total protein

  • Include appropriate negative controls (non-specific IgG) and positive controls

  • Confirm successful precipitation by Western blot analysis

• Potential interacting partners to investigate:

  • Other protocadherin family members

  • Cytoskeletal regulatory proteins

  • Signaling molecules like those in the PI3K/Akt pathway, which has been shown to interact with related protocadherins

The methodology used for Co-IP studies with related protocadherins can serve as a model. For example, studies with PCDH10 employed a Co-IP kit (Life Technologies) according to manufacturer's protocol, with subsequent analysis by Western blot to detect interactions with PI3K p85 and other signaling molecules .

How do PCDHA10 antibodies perform in multiplexed immunoassays?

Multiplexed detection of PCDHA10 alongside other proteins requires careful optimization:

• Antibody compatibility considerations:

  • Species cross-reactivity must be minimized when using multiple primary antibodies

  • When using rabbit polyclonal anti-PCDHA10 with other antibodies, select those raised in different host species (mouse, goat, etc.) to avoid cross-reactivity

  • For HRP-conjugated antibodies, consider spectral overlap when using multiple enzyme-based detection systems

• Multiplex immunofluorescence approach:

  • For co-localization studies, PCDHA10 antibodies have been successfully used alongside:

    • PECAM-1 antibodies (goat anti-PECAM-1, sc-1506, 1:500 dilution)

    • Alpha smooth muscle actin antibodies (mouse anti-α-SMA, ab7817, 1:500 dilution)

  • Secondary antibodies must be carefully selected to avoid cross-reactivity

• Sequential detection strategies:

  • For Western blot multiplexing, consider:

    • Stripping and reprobing membranes (may reduce sensitivity)

    • Using primary antibodies from different species with species-specific secondary antibodies

    • Employing antibodies that detect proteins of sufficiently different molecular weights

• Controls for multiplexed assays:

  • Single-antibody controls to establish baseline signals

  • Secondary-only controls to detect non-specific binding

  • Absorption controls with blocking peptides where available

When planning multiplexed experiments, it's essential to validate each antibody individually before combining them to ensure specificity and appropriate working dilutions in the experimental system of interest.

What approaches can be used to investigate PCDHA10's role in signaling pathways?

Investigating PCDHA10's role in signaling pathways requires multiple complementary approaches:

• Overexpression and knockdown studies:

  • Transfect cells with plasmids expressing PCDHA10 (e.g., pcDNA3.1-PCDHA10)

  • Use PCDHA10-specific siRNA (e.g., sc-106367) for knockdown studies

  • Analyze changes in downstream signaling molecules via Western blot

• Pathway analysis methodologies:

  • Based on studies of related protocadherins like PCDH10, examine:

    • PI3K/Akt signaling pathway components (p-AKT, p-MDM2)

    • Cell cycle regulators (p53, p21, cyclin D1)

    • Apoptosis markers (caspase-3, Bax, Bcl-2)

  • Western blot analysis starting with 20-50 μg total protein per lane

  • Use appropriate loading controls and phosphorylation-specific antibodies

• Co-immunoprecipitation to identify binding partners:

  • Immunoprecipitate with PCDHA10 antibodies and probe for potential signaling partners

  • Perform reciprocal Co-IP experiments to confirm interactions

  • Consider investigating PI3K p85 subunit interactions, as these have been documented with related protocadherins

• Functional readouts to assess pathway impact:

  • Cell proliferation assays (e.g., CCK-8)

  • Apoptosis detection (flow cytometry with Annexin V/PI staining)

  • Colony formation assays to assess long-term effects on cell growth and survival

Research on related protocadherins suggests that these proteins may function as tumor suppressors by modulating key signaling pathways. For example, PCDH10 has been shown to inhibit the PI3K/Akt signaling pathway, leading to reduced cell proliferation and increased apoptosis in hepatocellular carcinoma cells .

What are common troubleshooting strategies for weak or absent signals when using PCDHA10 HRP-conjugated antibodies?

When encountering weak or absent signals with PCDHA10 HRP-conjugated antibodies, consider these troubleshooting approaches:

• Sample preparation optimization:

  • Ensure complete cell lysis with appropriate buffer (RIPA for Western blot)

  • Include fresh protease inhibitors to prevent degradation

  • For tissue samples, optimize fixation and antigen retrieval methods

• Antibody concentration adjustments:

  • For Western blotting, try increasing antibody concentration (up to 1:500 dilution)

  • For immunofluorescence, higher concentrations may be required (starting at 1:50)

  • Extended incubation times (overnight at 4°C) may improve weak signals

• Detection system enhancement:

  • Use high-sensitivity ECL substrates for HRP detection

  • For particularly low abundance targets, consider amplification systems

  • Optimize exposure times based on signal strength

• Expression level verification:

  • Confirm PCDHA10 expression in your samples using alternative methods (qPCR)

  • Consider using positive control samples (e.g., U-87MG cells)

  • Test antibody performance in overexpression systems if endogenous levels are low

• Buffer and blocking optimization:

  • Test alternative blocking agents (BSA vs. milk)

  • Reduce washing stringency if signal is weak

  • Ensure optimal pH and salt concentration in all buffers

If signals remain weak after these optimizations, consider testing alternative antibodies or amplifying target expression through overexpression systems to validate the detection methodology.

How can cross-reactivity with other protocadherin family members be assessed and minimized?

The protocadherin family contains multiple members with sequence homology, making cross-reactivity a significant concern:

• Specificity validation strategies:

  • Peptide competition assays using the specific peptide against which the antibody was raised

  • Testing in knockout or knockdown systems where PCDHA10 expression is specifically reduced

  • Comparing detection patterns across multiple PCDHA10 antibodies recognizing different epitopes

• Epitope analysis:

  • Review the specific epitope recognized by the antibody (typically an internal region of PCDHA10)

  • Compare sequence homology with other protocadherin family members in this region

  • Consider custom antibodies against unique regions if cross-reactivity is problematic

• Pre-absorption controls:

  • Incubate the antibody with excess blocking peptide (e.g., sc-109747 P)

  • Compare signal with and without pre-absorption to identify non-specific binding

  • Include closely related family members in pre-absorption tests

• Experimental design considerations:

  • Include appropriate negative controls (tissues/cells not expressing PCDHA10)

  • When possible, confirm findings with orthogonal methods (qPCR, mass spectrometry)

  • Consider the biological context when interpreting results, as multiple protocadherins may be co-expressed

For PCDHA10 (S-12) antibody, the manufacturer notes it is raised against a peptide mapping within an internal region of PCDHA10 of mouse origin , which should be evaluated for potential cross-reactivity with other protocadherin family members in your experimental system.

What methodological adaptations are needed when using PCDHA10 antibodies for different species?

Species reactivity is a critical consideration when selecting PCDHA10 antibodies:

• Species reactivity profiles:

  • PCDHA10 Polyclonal Antibody (E-AB-91726) shows reactivity with human PCDHA10

  • PCDHA10 (S-12) is raised against mouse PCDHA10

  • Always verify species cross-reactivity before use in experimental systems

• Sequence homology considerations:

  • Compare the antibody epitope sequence with the corresponding region in your species of interest

  • Higher homology increases likelihood of cross-reactivity

  • For conserved regions, antibodies may recognize orthologs across multiple species

• Validation in target species:

  • Perform initial validation in positive control samples from the target species

  • Compare with known expression patterns from transcriptomic data

  • Consider using recombinant proteins of the target species as positive controls

• Protocol adaptations for different species:

  • Optimize antibody concentration for each species (typically higher dilutions for the original target species)

  • Adjust incubation conditions and blocking reagents based on target tissue

  • For immunohistochemistry applications, species-specific antigen retrieval methods may be required

For experiments in species not explicitly validated by manufacturers, preliminary validation steps are essential before proceeding with full experimental protocols.

How can I determine the optimal fixation methods for immunohistochemistry with PCDHA10 antibodies?

Optimization of fixation protocols is essential for successful immunohistochemistry with PCDHA10 antibodies:

• Fixative comparison:

  • Test paraformaldehyde (4%) fixation, which has been used successfully for embryonic tissue immunostaining with alpha-protocadherin antibodies

  • Compare with alternative fixatives (methanol, acetone, or combinations)

  • Optimize fixation duration (typically 15 minutes to 24 hours depending on sample size)

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Enzymatic retrieval (proteinase K, trypsin)

  • Optimize retrieval duration and temperature based on tissue type

• Protocol optimization:

  • Compare cryopreservation (as used for embryonic heart tissue ) versus paraffin embedding

  • For frozen sections, optimize section thickness (typically 5-10 μm)

  • Test permeabilization conditions to ensure antibody access to target epitopes

• Controls and validation:

  • Include positive control tissues with known PCDHA10 expression

  • Compare staining patterns with published expression data

  • Include blocking peptide controls to confirm specificity

Published protocols for immunostaining mouse embryonic heart tissue used 4% paraformaldehyde fixation followed by cryoembedding and sectioning. The alpha-protocadherin antibody (Synaptic Systems, 1:500) was used alongside PECAM-1 and alpha smooth muscle actin antibodies, with appropriate secondary antibodies for detection .

How can PCDHA10 antibodies be used to investigate its role in neurological development and disease?

PCDHA10, as a member of the protocadherin alpha gene cluster, has significant implications for neurological research:

• Developmental expression analysis:

  • Track PCDHA10 expression during critical neurodevelopmental periods

  • Use immunohistochemistry with PCDHA10 antibodies to map expression patterns in developing neural tissues

  • Compare with other protocadherin family members to identify unique and overlapping functions

• Synapse formation and maintenance studies:

  • Examine PCDHA10 localization at synaptic junctions using high-resolution microscopy

  • Investigate co-localization with synaptic markers

  • Quantify changes in PCDHA10 expression during synaptogenesis and in response to activity

• Disease-relevant applications:

  • Compare PCDHA10 expression in neurological disorder models

  • Investigate potential alterations in PCDHA10 expression or localization in:

    • Neurodevelopmental disorders

    • Neurodegenerative conditions

    • Brain tumor samples

• Functional manipulation approaches:

  • Use PCDHA10 antibodies for function-blocking studies in neuronal cultures

  • Combine with PCDHA10 knockdown using siRNA (e.g., sc-106367)

  • Assess effects on neuronal morphology, connectivity, and electrophysiological properties

Neural cadherin-like cell adhesion proteins such as PCDHA10 likely play critical roles in establishing and maintaining specific cell-cell connections in the brain . Antibodies against these proteins provide valuable tools for investigating these functions in normal development and disease states.