PKP2 Antibody, Biotin conjugated

What is the molecular weight of PKP2 protein, and how does this affect antibody detection?

PKP2 typically appears at approximately 97 kDa on Western blots, consistent with its calculated molecular weight of 97,415 Da . When using PKP2 antibodies, researchers should validate that their detected band appears at this expected size. Some variation might occur due to post-translational modifications or different isoforms. For optimal detection via Western blot, most protocols recommend antibody concentrations between 0.1-0.5 μg/ml .

Which tissue types and cell lines serve as positive controls for PKP2 antibody validation?

Based on validated experimental data, the following samples serve as reliable positive controls for PKP2 antibody testing:

These controls have demonstrated consistent PKP2 expression at the expected molecular weight of 97 kDa .

What are the recommended storage conditions for biotin-conjugated PKP2 antibodies?

Biotin-conjugated PKP2 antibodies should be aliquoted and stored at -20°C to maintain stability. Researchers should avoid repeated freeze-thaw cycles and minimize exposure to light, as biotin conjugates are light-sensitive . For long-term experiments requiring multiple uses of the same antibody preparation, creating multiple small aliquots during initial receipt is strongly recommended to preserve antibody functionality.

What protocol modifications are needed when using PKP2 antibodies across different experimental applications?

While PKP2 antibodies can be used in multiple applications, protocol optimization varies by technique:

• Western blot: Dilution range of 0.1-0.5 μg/ml is typically suitable for detecting PKP2 in human, rat, and mouse samples

• ELISA: Biotin-conjugated antibodies provide enhanced signal amplification through avidin/streptavidin systems. Optimal dilutions should be determined experimentally for each specific assay

• Immunohistochemistry: Antibody effectiveness in IHC may require tissue-specific optimization, particularly with cardiac tissues

How can cross-reactivity issues with PKP2 antibodies be addressed in multi-species studies?

Cross-reactivity evaluation is critical when applying PKP2 antibodies across species. While some antibodies are verified for human, mouse, and rat reactivity , cross-reactivity with other species (such as canine samples) requires validation. As noted in customer communications, researchers successfully using PKP2 antibodies in mouse tissues have inquired about potential reactivity with dog tissues .

When conducting multi-species studies:

• Always perform preliminary validation experiments with known positive controls

• Include negative controls lacking primary antibody

• Consider sequence homology between species when selecting antibodies

• Validate antibody specificity through Western blotting before proceeding to other applications

How can PKP2 antibodies be utilized to investigate connections between PKP2 expression and inflammatory pathways?

Recent research has revealed that PKP2 deficiency in cardiac myocytes correlates with upregulation of transcripts associated with inflammatory and immune responses . When designing experiments to investigate this connection:

• Use PKP2 antibodies in combination with markers of inflammation

• Implement co-localization studies to assess spatial relationships between PKP2 and inflammatory mediators

• Design time-course experiments to track changes in PKP2 expression during inflammation progression

Transcriptomic analysis has shown that PKP2 transcript abundance is endogenously linked to transcripts participating in inflammatory pathways, even in the absence of exogenous triggers . This suggests PKP2 antibodies can be valuable tools for investigating "sterile myocarditis" mechanisms.

What methodologies are most effective for studying desmosomal protein interactions involving PKP2?

For investigating protein-protein interactions involving PKP2:

• Co-immunoprecipitation: Using biotin-conjugated PKP2 antibodies with streptavidin beads can provide cleaner pull-downs with reduced background

• Proximity ligation assays: For detecting in situ protein interactions between PKP2 and other desmosomal components

• FRET-based approaches: For studying dynamic interactions in live cells

Research has demonstrated that PKP2 interacts with multiple desmosomal proteins including Desmoplakin, Plakoglobin, and Desmoglein 1 . When studying these interactions, consider using antibodies targeting specific domains of PKP2, as the protein has both desmosomal and nuclear functions.

What are the technical considerations when analyzing PKP2 expression in cardiac tissue from patients with arrhythmogenic cardiomyopathy?

IHC staining of myocardial samples has shown lower expression of PKP2, DSG2, and DSC2 in patients with arrhythmogenic cardiomyopathy compared to controls . When analyzing such samples:

• Use appropriate tissue controls from non-cardiac death subjects

• Implement quantitative scoring systems (normal, reduced, or absent immunoreactivity)

• Have multiple experienced pathologists independently score samples

• Consider myocardial region variability (septum vs. ventricular walls)

The pattern of PKP2 distribution in cardiac tissue is critical for diagnosis. In normal heart tissue, PKP2 localizes to intercalated discs, while in arrhythmogenic cardiomyopathy, this pattern is often disrupted or reduced .

How can researchers address non-specific binding when using PKP2 antibodies in Western blot applications?

To minimize non-specific binding:

• Optimize blocking conditions (BSA concentration, blocking time)

• Adjust antibody concentration (start with 0.1-0.5 μg/ml for Western blot)

• Increase washing stringency with higher salt concentrations or detergent

• Consider using biotin-conjugated secondary detection systems for enhanced signal-to-noise ratio

The specificity of PKP2 antibodies has been validated across multiple tissue types and cell lines, with consistent detection at 97 kDa , suggesting that non-specific binding can be minimized with proper protocol optimization.

What approaches can resolve discrepancies between PKP2 protein expression and transcript levels?

Researchers often encounter situations where protein and transcript levels of PKP2 do not correlate. This can be addressed by:

• Performing parallel analyses of protein (Western blot) and mRNA (qPCR)

• Using RiboTag approaches to specifically analyze the ribosome-resident transcriptome of cardiomyocytes

• Implementing pulse-chase experiments to assess PKP2 protein stability and turnover

• Investigating post-transcriptional regulatory mechanisms

Recent research using cardiac-specific, tamoxifen-activated PKP2-knockout mice crossed with RiboTag lines has helped characterize the complex relationship between PKP2 transcripts and protein expression , providing methodological frameworks for addressing such discrepancies.

How can PKP2 antibodies contribute to understanding the mechanistic link between desmosomes and inflammatory signaling?

Transcriptomic coupling between PKP2 and inflammatory/immune response genes suggests novel research directions :

• Use PKP2 antibodies to track subcellular localization changes during inflammatory activation

• Implement ChIP-seq approaches with biotin-conjugated PKP2 antibodies to identify potential transcriptional regulatory roles

• Design cell type-specific assays to distinguish PKP2 function in cardiomyocytes versus immune cells

• Develop co-culture systems to investigate cell-cell communication mediated by PKP2

Research has identified transcriptomic pathways linking PKP2 deficiency to responses typically associated with viral or bacterial infections, particularly those known to produce myocarditis , suggesting PKP2 antibodies could be valuable tools for investigating non-infectious inflammatory cardiomyopathies.

What are the considerations for using PKP2 antibodies in studies of digenic inheritance patterns in arrhythmogenic cardiomyopathy?

When investigating patients with multiple desmosomal gene variants:

• Design multiplex immunostaining protocols using differently labeled antibodies against PKP2 and other desmosomal proteins

• Implement quantitative image analysis to assess co-localization patterns

• Compare protein expression patterns between patients with isolated PKP2 mutations versus those with digenic variants