PKP2 Antibody, HRP conjugated

What is PKP2 and why is it significant in cardiovascular research?

Plakophilin-2 (PKP2) is an armadillo protein that serves as a critical regulatory component in the cytoplasmic plaques of various cell-cell junctions, particularly in the composite junctions (areae compositae) of myocardiac intercalated disks . PKP2 is the most frequently affected gene in arrhythmogenic cardiomyopathy (ACM), a severe cardiac disorder characterized by lethal arrhythmias and sudden cardiac death . Its significance in cardiovascular research stems from its essential role in maintaining cardiac structural integrity, with PKP2 knockout in embryonic hearts resulting in dispersed desmoplakin aggregates, disrupted myocardial structure, and cardiac death . Additionally, PKP2 interacts with α-T-catenin and influences connexin-43 function in gap junctions, highlighting its importance in cardiac conduction .

What cellular localizations can be detected using PKP2 antibodies?

High-sensitivity PKP2 antibodies can detect the protein in multiple cellular locations:

• Cytoplasmic plaques of desmosomal junctions

• Composite junctions of myocardial intercalated disks

• Non-desmosomal cell-cell junction structures (puncta adhaerentia and fasciae adhaerentes)

• Nucleoplasmic protein complexes (showing granular appearance while leaving nucleoli negative)

• Heterotypic desmosomes (particularly in the neuroendocrine cell half)

This dual localization pattern (junctional and nuclear) is particularly important in proliferative non-epithelial cells, cardiac interstitial cells, and cardiac myxomata .

How do I select the appropriate PKP2 antibody for detecting specific isoforms?

When selecting antibodies for specific PKP2 isoform detection, consider:

For comprehensive detection of both isoforms, select antibodies targeting conserved regions. The specific monoclonal and polyclonal antibodies described by Schmidt et al. provided similar results in immunoblotting of SDS-PAGE-separated polypeptides from normal and tumor tissue samples, including vertebrate heart tissue and cell cultures . When isoform specificity is critical, validate with recombinant proteins representing each isoform variant.

What are the optimal fixation and antigen retrieval methods for PKP2 immunohistochemistry?

For optimal PKP2 detection in tissue samples:

Fixation Protocol:

• Formaldehyde fixation (4% paraformaldehyde) is compatible with PKP2 antibody detection

• Paraffin-embedded tissue samples have been successfully used with high-specificity PKP2 antibodies

Antigen Retrieval Recommendations:

• Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

• For cardiac tissues, add a mild protease digestion step to enhance antibody accessibility to intercalated disks

• For dual localization studies (detecting both junctional and nuclear PKP2), optimize retrieval conditions to preserve both pools

These methods have been validated for detection of both junctional and nuclear PKP2 forms, showing the characteristic granular nucleoplasmic appearance with negative nucleoli .

How should I optimize Western blot protocols for PKP2 detection?

For optimal Western blot detection of PKP2:

• Sample Preparation:

  • Use RIPA buffer with protease inhibitors for total protein extraction

  • For membrane-associated PKP2, consider mild detergent conditions

  • For nuclear PKP2, employ nuclear fractionation protocols

• Gel Electrophoresis Parameters:

  • 7.5-10% SDS-PAGE gels provide optimal resolution

  • Transfer to PVDF membrane at 100V for 60 minutes

• PKP2 Antibody Conditions:

  • Primary antibody dilution: 1:1000-1:2000 (optimize based on antibody concentration)

  • Incubation: Overnight at 4°C for enhanced sensitivity

  • HRP-conjugated antibody: Use directly at manufacturer's recommended dilution

  • For non-conjugated primary antibodies: Follow with HRP-secondary at 1:5000 dilution

• Expected Results:

  • Full-length PKP2: ~100 kDa band

  • Potential isoforms or processed forms may appear as additional bands

  • Verify specificity with positive controls (heart tissue extracts)

How can PKP2 antibodies be used to investigate arrhythmogenic cardiomyopathy mechanisms?

PKP2 antibodies are instrumental in investigating ACM mechanisms through multiple approaches:

• Mutation Impact Assessment:

  • Western blot quantification of PKP2 levels in patient-derived samples or disease models

  • Immunofluorescence localization to assess PKP2 trafficking and junctional incorporation

• Therapeutic Monitoring in Gene Therapy Studies:

  • Tracking PKP2 restoration in AAV-mediated delivery models

  • Recent research demonstrated that AAV9-PKP2 treatment prevented cardiac dysfunction in heterozygous PKP2 c.1755delA/WT mice, with antibodies being essential for confirming protein restoration

• Interactome Analysis:

  • Co-immunoprecipitation with PKP2 antibodies to identify binding partners

  • PKP2 antibodies helped identify that PKP2 restoration improved levels of other junctional proteins decreased due to mutations

• Functional Recovery Validation:

  • Immunofluorescence to confirm desmosomal integrity recovery after therapeutic intervention

  • Antibody-based detection showing PKP2 restoration can enhance contractile function and normalize contraction kinetics in PKP2 mutant engineered human myocardium

What methodological approaches can distinguish between junctional and nuclear PKP2?

To differentiate between junctional and nuclear PKP2 pools:

Fractionation Approach:

• Perform subcellular fractionation to separate membrane, cytosolic, and nuclear fractions

• Use Western blotting with PKP2 antibodies on each fraction

• Confirm fraction purity using markers: E-cadherin (membrane), GAPDH (cytosolic), Lamin B1 (nuclear)

Immunofluorescence Method:

• Double immunostaining with PKP2 antibody and markers for:

  • Desmosomes (desmoplakin, desmoglein)

  • Adherens junctions (β-catenin)

  • Nuclear envelope (lamin B1)

• Confocal microscopy with z-stack analysis to confirm localization

• Use line scan analysis to quantify relative distribution

Studies have shown that PKP2 antibodies can detect both junction-bound and nuclear forms, which appear as granular nucleoplasmic structures that exclude nucleoli . This dual localization is particularly evident in highly proliferative non-epithelial cells, cardiac interstitial cells, and cardiac myxomata .

How can PKP2 antibodies be utilized in iPSC-derived cardiomyocyte disease models?

PKP2 antibodies are essential tools for iPSC-derived cardiomyocyte disease models:

• Disease Modeling Validation:

  • Confirm PKP2 haploinsufficiency in iPSC-CMs with PKP2 mutations

  • Recent research used PKP2 antibodies to validate PKP2 c.2013delC/WT induced pluripotent stem cell-derived cardiomyocytes

• Therapeutic Response Assessment:

  • Quantify PKP2 restoration after gene therapy interventions

  • Monitor corresponding changes in other junctional proteins

  • Research showed that AAV-mediated delivery of PKP2 in mutant iPSC-CMs restored not only PKP2 levels but also other junctional proteins decreased due to the mutation

• Functional Correlation Studies:

  • Combine PKP2 immunostaining with functional measurements

  • PKP2 restoration was shown to improve sodium conduction, indicating rescue of arrhythmic substrate in PKP2 mutant iPSC-CMs

• Protocol Modifications for iPSC-CMs:

  • Fix cells with 4% PFA for 15 minutes at room temperature

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

  • Block with 5% BSA/PBS for 1 hour

  • Use PKP2 antibody at 1:100-1:200 dilution

How do I validate PKP2 antibody specificity and performance?

To ensure antibody specificity and performance:

• Positive Controls:

  • Heart tissue sections (particularly intercalated disks)

  • Cell lines with confirmed PKP2 expression (e.g., cardiac fibroblasts)

  • PKP2 overexpression systems

• Negative Controls:

  • PKP2 knockout or knockdown samples

  • Primary antibody omission controls

  • Isotype control antibodies

• Specificity Tests:

  • Pre-absorption with immunizing peptide

  • Western blot showing single band at expected molecular weight (~100 kDa)

  • Parallel testing with multiple PKP2 antibodies targeting different epitopes

• Methodological Validation:

  • Systematic antibody dilution tests

  • Multiple fixation method comparisons

  • Cross-validation between immunofluorescence and Western blot results

Research has demonstrated that specific monoclonal and polyclonal PKP2 antibodies can provide consistent results across different applications, including Western blot of SDS-PAGE-separated polypeptides from normal and tumor tissue samples .

What are common pitfalls when using PKP2-HRP conjugated antibodies and how can they be addressed?

Common pitfalls and solutions when using PKP2-HRP conjugated antibodies:

For direct HRP-conjugated antibodies, be particularly mindful of storage conditions, as conjugated antibodies can lose activity more rapidly than non-conjugated ones. Store small aliquots at -20°C with stabilizing proteins.

How can PKP2 antibodies help investigate PKP2's role in viral defense mechanisms?

PKP2 antibodies are valuable tools for exploring PKP2's unexpected role in viral defense:

• Virus-Host Protein Interaction Studies:

  • Use PKP2 antibodies for co-immunoprecipitation with viral proteins

  • Research has shown that PKP2 interacts with the PB1 protein of influenza A virus (IAV)

• Restriction Mechanism Analysis:

  • Immunofluorescence to track PKP2 localization during viral infection

  • Western blot to quantify PKP2 expression changes during infection

  • Research demonstrated that PKP2 restricts IAV replication by competing with PB2 for PB1 binding, thereby perturbing the IAV polymerase complex

• Experimental Approach:

  • Use PKP2 antibodies to isolate protein complexes from infected cells

  • Compare PKP2 interaction partners between infected and uninfected cells

  • Assess PKP2 levels in various cellular compartments during infection

This research direction was established through comparative analyses of influenza-host protein interactomes, which identified PKP2 as a natural inhibitor of the IAV polymerase complex . PKP2 antibodies were crucial for mapping these protein-protein interactions.

How can PKP2 antibodies be used to study its role in endothelial permeability and inflammation?

PKP2 antibodies provide valuable insights into PKP2's role in regulating endothelial permeability:

• Expression Analysis in Inflammatory Conditions:

  • Western blot and immunofluorescence to quantify PKP2 in response to inflammatory stimuli

  • Recent research showed increased PKP2 expression in pancreatic tissues of severe acute pancreatitis (SAP) mice and in human umbilical vein endothelial cells (HUVECs) after lipopolysaccharide stimulation

• Mechanistic Studies:

  • Co-immunoprecipitation with PKP2 antibodies to identify junctional protein interactions

  • PKP2 overexpression was shown to increase levels of tight junction proteins (ZO-1, occludin, claudin1) and adherens junction proteins (β-catenin)

• Signaling Pathway Analysis:

  • Western blot to assess phosphorylation status of pathway components

  • Research demonstrated that PKP2 overexpression counteracted the inhibitory effect of SB203580 (a p38/MAPK signaling pathway inhibitor), restoring ZO-1, β-catenin, and claudin1 levels

• Functional Correlation Studies:

  • Combine PKP2 immunostaining with permeability assays

  • PKP2 overexpression was shown to reduce endothelial cell permeability and improve cytoskeleton relaxation in response to acute inflammatory stimulation

Research indicated that the PPARγ activator rosiglitazone upregulates PKP2 expression and reduces inflammation-induced permeability, suggesting a potential therapeutic approach .

What considerations are important when using PKP2 antibodies in studies of cardiac development?

When using PKP2 antibodies to study cardiac development:

• Developmental Stage-Specific Protocols:

  • Adjust fixation times for embryonic and fetal tissues (typically shorter)

  • Optimize antigen retrieval for developing cardiac structures

  • Consider tissue clearing techniques for whole-mount imaging

• Co-localization Studies:

  • Pair PKP2 antibodies with developmental markers:

    • Cardiac progenitor markers (Nkx2.5, GATA4)

    • Differentiation markers (cTnT, α-actinin)

    • Other junctional proteins as they appear developmentally

• Technical Adaptations:

  • For embryonic tissues: Use lower antibody concentrations to reduce background

  • For small samples: Consider whole-mount immunostaining

  • For quantitative analysis: Include stage-matched wild-type controls

• Interpretation Challenges:

  • PKP2 junctional localization changes during development

  • Nuclear PKP2 may be more prominent in early developmental stages

  • PKP2 levels in cardiac valvular interstitial cells during fetal development should be carefully evaluated

Research has demonstrated that PKP2 knockout in embryonic hearts leads to dispersed desmoplakin aggregates, highlighting its critical role in cardiac development . Additionally, studies on cultured cardiomyocytes have shown that PKP2 knockdown during early development can result in junction splitting and dissociation .