PPA2 Antibody, HRP conjugated

What species reactivity do commercially available PPA2 antibodies demonstrate?

Commercial PPA2 antibodies show reactivity with multiple species, with most validated for human, mouse, and rat samples. For example, the rabbit recombinant monoclonal antibody clone EPR13086 has been experimentally validated for all three species , while other clones like EPR13087(B) have been primarily validated for human samples . When planning experiments with samples from less common research species, it's advisable to check sequence homology predictions or perform preliminary validation testing to confirm antibody compatibility.

What applications are PPA2 antibodies validated for?

PPA2 antibodies are validated for multiple research applications. The EPR13086 clone has been tested and confirmed suitable for Western blot (WB), immunohistochemistry on paraffin-embedded sections (IHC-P), and immunoprecipitation (IP) across human, mouse, and rat samples . The EPR13087(B) clone is validated for Western blot, immunoprecipitation, and flow cytometry (intracellular) specifically with human samples . When selecting an HRP-conjugated variant, these application validations remain relevant, though the conjugation may optimize performance specifically for detection-based methods like Western blotting and ELISA.

What are the recommended dilution parameters for PPA2 antibodies in Western blot?

For optimal Western blot results, PPA2 antibodies typically require specific dilution ratios depending on the antibody clone and sample type. The EPR13086 clone has been validated at 1:1000 dilution for heart tissue lysates and at 1:10000 dilution for HepG2 whole cell lysates . The EPR13087(B) clone is recommended at 1:1000 dilution for various sample types including human fetal liver tissue lysate, HepG2 cell lysate, human fetal kidney tissue lysate, U-87 MG cell lysate, and 293T cell lysate . When using HRP-conjugated variants, these dilution parameters provide a starting point, though optimization may be required based on the conjugation efficiency and detection method.

What explains the discrepancy between predicted and observed molecular weights for PPA2?

A notable discrepancy exists between the predicted molecular weight of PPA2 (38 kDa) and its observed migration pattern in SDS-PAGE (approximately 35 kDa) . This difference is consistently observed across multiple antibody clones and sample types, suggesting it reflects an intrinsic property of the PPA2 protein rather than an antibody-specific artifact. Possible explanations include post-translational modifications, anomalous migration due to protein structure, or proteolytic processing during sample preparation. Researchers should note this characteristic migration pattern when evaluating Western blot results to avoid misinterpreting the 35 kDa band as non-specific binding.

What blocking and dilution buffers optimize PPA2 antibody performance?

For optimal PPA2 antibody performance in Western blot applications, 5% non-fat dry milk in TBST (5% NFDM/TBST) has been experimentally validated as an effective blocking and dilution buffer . This combination minimizes background while maintaining specific signal intensity. For HRP-conjugated antibodies, this buffer composition remains appropriate, though care should be taken to ensure the blocking agent doesn't interfere with HRP activity. When troubleshooting high background or weak signals, alternative blocking agents such as BSA may be tested, but the validated milk-based buffer provides a reliable starting point for most experimental systems.

How can PPA2 antibodies be used to investigate mitochondrial function?

PPA2 antibodies provide valuable tools for investigating mitochondrial function and dysfunction, as PPA2 activity is essential for proper mitochondrial membrane potential and organization . Research approaches include:

• Comparative expression analysis in normal versus disease model tissues using Western blotting or IHC

• Co-localization studies with other mitochondrial markers using immunofluorescence

• Analysis of PPA2 expression changes during mitochondrial stress responses

• Correlation of PPA2 levels with functional mitochondrial parameters

HRP-conjugated PPA2 antibodies offer advantages in Western blot detection due to their direct enzymatic signal generation, potentially enhancing sensitivity when studying subtle changes in PPA2 expression under different mitochondrial conditions.

What methodological approaches can resolve contradictory PPA2 expression data?

When facing contradictory PPA2 expression data across different experimental techniques, implement this systematic reconciliation approach:

• Antibody validation:

  • Verify epitope specificity across all antibodies used

  • Test multiple antibodies targeting different PPA2 epitopes (e.g., EPR13086 and EPR13087(B))

  • Confirm all antibodies detect the same PPA2 isoform

• Technical considerations:

  • Evaluate sample preparation differences that might affect epitope accessibility

  • Consider the impact of different blocking buffers on antibody performance

  • Account for the discrepancy between predicted (38 kDa) and observed (35 kDa) molecular weights

• Biological variables:

  • Assess cell/tissue heterogeneity effects on bulk versus single-cell measurements

  • Consider post-translational modifications that may affect detection

  • Evaluate subcellular fractionation quality when analyzing mitochondrial proteins

What advantages do HRP-conjugated PPA2 antibodies offer over unconjugated versions?

HRP-conjugated PPA2 antibodies offer several methodological advantages:

• Streamlined protocols: Elimination of secondary antibody incubation and washing steps reduces experiment time and potential for error

• Enhanced sensitivity: Direct conjugation can improve signal-to-noise ratio in samples with low PPA2 abundance

• Reduced cross-reactivity: Elimination of secondary antibodies minimizes species cross-reactivity issues

• Consistent signal generation: Lower batch-to-batch variation compared to two-step detection systems

• Cost efficiency: Long-term usage reduces reagent costs by eliminating secondary antibody requirements

These advantages are particularly beneficial when working with sample types where PPA2 is expressed at low levels or when performing high-throughput analyses.

How should researchers optimize detection methods for HRP-conjugated PPA2 antibodies?

Optimization of detection methods for HRP-conjugated PPA2 antibodies should consider:

• Substrate selection:

  • Enhanced chemiluminescence (ECL) offers excellent sensitivity for detecting low abundance PPA2

  • Chromogenic substrates (DAB, TMB) provide permanent signals but with lower sensitivity

  • Chemifluorescent substrates balance sensitivity with signal stability

• Exposure parameters:

  • Multiple exposure times should be tested to capture the optimal signal-to-noise ratio

  • Digital imaging systems allow real-time monitoring of signal development

  • For quantitative analyses, verify linearity of signal within exposure range

• Background reduction:

  • Optimize blocking conditions (5% NFDM/TBST has been validated)

  • Include appropriate detergents in wash buffers

  • Consider using specialized low-background membranes

• Control implementation:

  • Include positive controls with known PPA2 expression (e.g., HepG2 cells, heart tissue)

  • Run negative controls to assess non-specific binding

What troubleshooting approaches address common issues with HRP-conjugated PPA2 antibodies?

When encountering problems with HRP-conjugated PPA2 antibody performance, implement these targeted troubleshooting strategies:

• No signal or weak signal:

  • Verify PPA2 expression in your sample type

  • Confirm HRP conjugate activity with substrate dot test

  • Increase antibody concentration or extend incubation time

  • Consider enhanced detection substrates

  • Validate expected band size (approximately 35 kDa rather than predicted 38 kDa)

• High background:

  • Increase blocking time and concentration

  • Add additional wash steps with fresh buffer

  • Reduce antibody concentration

  • Use alternative blocking agents if 5% NFDM/TBST is insufficient

  • Consider membrane optimization or pre-adsorption procedures

• Multiple bands:

  • Compare with known migration pattern (primary band at ~35 kDa)

  • Evaluate sample integrity (minimize protein degradation)

  • Consider post-translational modifications or isoforms

  • Validate specificity with knockdown/knockout controls

How can researchers design experiments to study PPA2 in mitochondrial pathology models?

To effectively study PPA2 in the context of mitochondrial pathology, implement this comprehensive experimental design:

• Model selection and validation:

  • Cell lines with established mitochondrial defects

  • Patient-derived samples with mitochondrial disorders

  • Animal models of mitochondrial dysfunction

  • PPA2 knockdown/knockout systems to establish causality

• Multi-parameter assessment:

  • PPA2 expression: Western blot with HRP-conjugated antibodies at validated dilutions (1:1000-1:10000)

  • Mitochondrial function: Membrane potential, respiration, ROS production

  • Mitochondrial morphology: Structural analysis via microscopy

  • Cell viability and stress markers: Correlate with PPA2 levels

• Experimental controls:

  • Wild-type comparison groups

  • Rescue experiments via PPA2 re-expression

  • Positive controls with known mitochondrial disruptors

  • Technical controls validating antibody specificity

• Analytical approaches:

  • Quantitative Western blot analysis (accounting for the 35 kDa observed band)

  • Statistical correlation between PPA2 levels and functional parameters

  • Longitudinal analysis of PPA2 changes during disease progression

What considerations are important when selecting between different PPA2 antibody clones?

Selection between available PPA2 antibody clones should be guided by these application-specific considerations:

For cross-species studies or multi-technique approaches, EPR13086 offers broader validated applications. For human cell line studies incorporating flow cytometry, EPR13087(B) provides specific advantages. When selecting an HRP-conjugated variant, consider which clone's epitope is less likely to be affected by the conjugation process.

How can researchers validate the specificity of PPA2 antibody signal in experimental systems?

A comprehensive validation strategy for PPA2 antibodies should include:

• Genetic approaches:

  • RNAi-mediated knockdown of PPA2 (siRNA or shRNA)

  • CRISPR/Cas9 knockout models

  • Correlation of protein signal reduction with mRNA depletion

• Biochemical validation:

  • Peptide competition assays

  • Analysis of multiple antibodies targeting different PPA2 epitopes

  • Immunoprecipitation followed by mass spectrometry

• Technical controls:

  • Omission of primary antibody

  • Isotype control antibodies

  • Secondary-only controls (for non-conjugated antibodies)

  • Substrate-only controls (for HRP-conjugated antibodies)

• Cross-technique validation:

  • Correlation between Western blot, IHC, and flow cytometry results

  • Subcellular fractionation to confirm mitochondrial localization

  • Co-localization with established mitochondrial markers

For HRP-conjugated PPA2 antibodies specifically, additional validation should include comparison with unconjugated versions to ensure conjugation doesn't alter specificity or binding characteristics.

How should PPA2 expression be quantified and normalized in Western blot analysis?

Accurate quantification of PPA2 requires careful consideration of normalization approaches:

• Loading control selection:

  • Mitochondrial-specific loading controls (e.g., VDAC, TOM20) are preferable when analyzing PPA2

  • Total protein normalization may provide more consistent results across diverse sample types

• Band identification:

  • Focus quantification on the established 35 kDa band rather than the predicted 38 kDa position

  • Verify single band specificity in your experimental system

• Quantification methodology:

  • Use densitometry software with background subtraction capabilities

  • Establish linear dynamic range using serial dilutions of a reference sample

  • Apply consistent analysis parameters across all experimental replicates

• Statistical analysis:

  • Analyze minimum of three biological replicates

  • Apply appropriate statistical tests based on experimental design

  • Consider log transformation for data with wide expression ranges

What approaches can reconcile discrepancies in PPA2 localization across different techniques?

When facing contradictory data regarding PPA2 localization, implement this systematic reconciliation approach:

• Antibody considerations:

  • Verify epitope accessibility in different preparation methods

  • Compare results from multiple PPA2 antibody clones (EPR13086, EPR13087(B))

  • Evaluate fixation effects on epitope recognition

• Technical validation:

  • Confirm subcellular fractionation purity with established markers

  • Evaluate fixation and permeabilization protocols for IHC and IF

  • Consider native versus denatured protein conformation effects

• Biological validation:

  • Correlate observed localization with known PPA2 function

  • Evaluate potential redistribution under experimental conditions

  • Consider development or tissue-specific expression patterns

• Resolution strategies:

  • Super-resolution microscopy for precise localization

  • Proximity ligation assays for protein interaction verification

  • Correlative light and electron microscopy for ultrastructural context

How can researchers integrate PPA2 expression data with functional mitochondrial parameters?

To meaningfully correlate PPA2 expression with mitochondrial function:

• Experimental design:

  • Parallel sampling for protein analysis and functional assays

  • Time-course studies to capture dynamic relationships

  • Perturbation experiments with graded PPA2 modulation

• Data integration approaches:

  • Correlation analysis between PPA2 levels and functional parameters

  • Multivariate analysis to identify complex relationships

  • Hierarchical clustering to identify co-regulated parameters

• Visualization methods:

  • Scatter plots with PPA2 expression versus functional readouts

  • Heat maps integrating multiple parameters

  • Principal component analysis to identify key variables

• Causal relationship testing:

  • Rescue experiments in PPA2-depleted systems

  • Dose-response relationships with PPA2 modulation

  • Kinetic analysis of changes following acute PPA2 inhibition

This integrated approach allows researchers to move beyond correlative observations to establish mechanistic connections between PPA2 expression and mitochondrial function in various physiological and pathological contexts.