PP2A15 Antibody

What is PP2A and why is it important in research?

PP2A (Protein Phosphatase 2A) is a critical serine/threonine phosphatase that regulates numerous cellular processes including cell cycle progression, signal transduction, and apoptosis. The protein is encoded by the gene PTPA (protein phosphatase 2 phosphatase activator) in humans, resulting in a 358-amino acid protein belonging to the PTPA-type PPIase family. Its cellular localization is both cytoplasmic and nuclear. PP2A has gained significant research interest due to its role as a tumor suppressor and its dysregulation in various cancers and neurodegenerative diseases . Understanding PP2A function through antibody-based detection methods is therefore essential for elucidating disease mechanisms and developing potential therapeutic approaches.

What applications are PP2A antibodies commonly used for?

PP2A antibodies are utilized in multiple experimental applications across molecular and cellular biology research. The most common applications include Western Blotting (WB) for protein expression quantification, Enzyme-Linked Immunosorbent Assays (ELISA) for protein detection in solutions, Flow Cytometry (FCM) for cellular analysis, Immunofluorescence (IF) for subcellular localization studies, and Immunohistochemistry (IHC) for tissue-level expression analysis . Each application requires specific validation parameters, and researchers should select antibodies that have been validated for their intended experimental application rather than assuming cross-application validity.

What is the difference between monoclonal and polyclonal PP2A antibodies?

Monoclonal PP2A antibodies (like clone E155 from Abcam and F-8 from Santa Cruz) are derived from a single B-cell clone and recognize a single epitope, offering high specificity but potentially lower sensitivity. Polyclonal antibodies (like those from R&D Systems) contain a mixture of immunoglobulins that recognize multiple epitopes, potentially providing higher sensitivity but with increased risk of cross-reactivity . For PP2A research, this distinction is particularly important when studying post-translational modifications, as some monoclonal antibodies have been shown to have unexpected binding characteristics, such as recognizing both phosphorylated and unphosphorylated forms despite being marketed as phospho-specific .

How should I validate a PP2A antibody before using it in my experiments?

Proper validation of PP2A antibodies requires a multi-step approach:

• Knockout/knockdown controls: Generate PP2A-null or knockdown models to confirm antibody specificity

• Phosphatase treatments: For phospho-specific antibodies, treat samples with phosphatases to confirm specificity

• Peptide competition assays: Use synthetic peptides of known modification status to verify epitope recognition

• Mass spectrometry validation: When possible, confirm antibody-detected modifications through MS analysis

• Multiple antibody comparison: Use different antibodies targeting distinct epitopes to confirm results

This comprehensive validation is particularly critical for PP2A given the documented issues with phospho-Tyr307 antibodies that cannot differentiate between phosphorylated and unphosphorylated forms of PP2Ac . Using phospho-incompetent mutants (e.g., Y307F) provides an excellent negative control for validating phospho-specific antibodies.

What controls should I include when using PP2A antibodies for detecting post-translational modifications?

When studying PP2A post-translational modifications, the following controls are essential:

• Phosphatase/kinase treatments: Treat cell lysates with appropriate enzymes to generate positive and negative controls

• Mutation controls: Express phospho-mimetic (e.g., Y307E) or phospho-incompetent (e.g., Y307F) mutants

• Stimulation controls: Use treatments known to induce or reduce the modification (e.g., EGF stimulation for Tyr307 phosphorylation)

• Competing peptides: Include modified and unmodified peptides to confirm specificity

• Alternative detection methods: Validate findings using mass spectrometry or other non-antibody methods

These controls are particularly important given that commonly used phospho-Tyr307 antibodies have been shown to detect unphosphorylated PP2Ac with equal or greater affinity than the phosphorylated form, leading to potentially misleading interpretations of experimental results .

How can I address cross-reactivity issues with PP2A antibodies?

Addressing cross-reactivity requires:

• Epitope mapping: Determine the exact sequence recognized by the antibody

• Pre-absorption: Incubate antibodies with purified recombinant proteins or peptides to remove cross-reactive antibodies

• Fractionation techniques: Analyze subcellular fractions separately to reduce background

• BLAST analysis: Identify potential cross-reactive proteins with similar epitopes

• Western blot optimization: Adjust blocking conditions, antibody dilutions, and washing procedures

For PP2A antibodies, it's particularly important to test for sensitivity to adjacent post-translational modifications. For example, the E155 and F-8 phospho-Tyr307 antibodies show significantly reduced binding when Leu309 is methylated, even though they are not marketed as methylation-sensitive .

How do different post-translational modifications affect PP2A antibody binding?

Post-translational modifications significantly impact PP2A antibody recognition in complex ways not typically described in product data sheets. Research has demonstrated that:

This complex interplay between modifications creates significant challenges in interpreting results. For example, an apparent increase in "phospho-Tyr307" signal using the R&D antibody could actually reflect increased Thr304 phosphorylation rather than Tyr307 phosphorylation . Researchers should consider comprehensive modification analysis using mass spectrometry to fully understand the PP2A modification state.

What are the implications of the recent reinterpretation of PP2A phospho-antibody specificity for cancer research?

The discovery that widely used phospho-Tyr307 antibodies cannot reliably distinguish between phosphorylated and unphosphorylated PP2A has profound implications for cancer research:

This situation exemplifies how insufficient antibody validation can lead to widespread misinterpretation in an entire research field, potentially misdirecting resources and therapeutic development efforts .

How can computational approaches improve PP2A antibody specificity design?

Advanced computational methods offer promising approaches to designing highly specific PP2A antibodies:

• Binding mode identification: Computational models can identify different binding modes associated with specific ligands or epitopes

• High-throughput sequencing analysis: Combining experimental phage display with computational analysis can enhance specificity prediction

• Specificity profile customization: Algorithms can design antibodies with tailored specificity profiles, targeting specific PP2A isoforms or modification states

• Cross-specificity engineering: Computational approaches can create antibodies that recognize multiple pre-defined targets while excluding others

• Bias mitigation: Computational methods can identify and correct for experimental artifacts and selection biases

These approaches are particularly valuable for creating antibodies that can reliably distinguish between highly similar epitopes, such as differently modified states of PP2A . The integration of experimental and computational methods represents the cutting edge of antibody engineering for challenging targets like PP2A.

What are the best practices for using PP2A antibodies in different experimental applications?

Each application requires specific optimization:

Western Blotting:

• Use multiple antibodies targeting different epitopes

• Include phosphatase/kinase treatment controls

• Optimize transfer conditions for this 36kDa protein

• Consider native vs. reducing conditions based on epitope accessibility

Immunoprecipitation:

• Verify antibody compatibility with IP buffer conditions

• Test for co-precipitation of PP2A regulatory subunits

• Consider native vs. denatured IP based on complex stability

Immunohistochemistry/Immunofluorescence:

• Optimize fixation methods (paraformaldehyde vs. methanol)

• Validate antibody performance in tissue-specific contexts

• Use antigen retrieval methods appropriate for phospho-epitopes

Flow Cytometry:

• Verify membrane permeabilization for this intracellular target

• Use appropriate blocking to reduce non-specific binding

• Include isotype controls specific to each antibody class

These application-specific considerations help maximize signal specificity and minimize artifacts that might lead to misinterpretation .

How can I effectively troubleshoot non-specific binding with PP2A antibodies?

Non-specific binding can be addressed through systematic troubleshooting:

• Increase blocking stringency: Use 5% BSA instead of milk for phospho-specific antibodies

• Adjust antibody concentration: Perform dilution series to identify optimal concentration

• Modify washing conditions: Increase wash duration or add detergents like Tween-20

• Implement epitope competition: Include synthetic peptides representing the target epitope

• Evaluate fixation impact: Test different fixation methods that may affect epitope accessibility

For PP2A specifically, consider that observed "non-specific" binding might actually reflect antibody sensitivity to unexpected modifications. For example, apparent cross-reactivity might be due to sensitivity to Thr304 phosphorylation or Leu309 methylation rather than true non-specificity .

What are the recommended approaches for multiplexing PP2A antibodies with other targets?

Effective multiplexing requires careful planning:

• Antibody species selection: Choose primary antibodies from different host species

• Fluorophore selection: Select fluorophores with minimal spectral overlap

• Sequential staining: Apply one antibody set, fix, then apply the second set

• Cross-reactivity testing: Validate that secondary antibodies don't cross-react

• Control for epitope masking: Ensure antibody binding doesn't block adjacent epitopes

For PP2A specifically, consider that regulatory subunits and interacting proteins may sterically hinder epitope accessibility. When multiplexing, validate that antibody combinations don't affect each other's binding efficiency through steric hindrance or epitope masking .

How should I interpret contradictory results obtained with different PP2A antibodies?

When facing contradictory results:

• Evaluate antibody validation: Review validation data for each antibody, particularly regarding specificity

• Consider epitope differences: Different antibodies may recognize distinct conformations or modification states

• Assess buffer compatibility: Some antibodies perform differently under varying experimental conditions

• Examine lot-to-lot variation: Request validation data specific to the antibody lot used

• Implement orthogonal methods: Use non-antibody methods (mass spectrometry, activity assays) to resolve contradictions

The documented issues with phospho-Tyr307 antibodies highlight that contradictions may reflect actual antibody limitations rather than experimental error. For example, different signals between antibodies might reflect differential sensitivity to modifications at Thr304 or Leu309 rather than differences in Tyr307 phosphorylation .

What strategies can improve reproducibility when using PP2A antibodies across different studies?

To enhance reproducibility:

• Detailed reporting: Document antibody catalog numbers, lot numbers, dilutions, and incubation conditions

• Validation sharing: Include antibody validation data in publications or supplements

• Multiple antibody approach: Use at least two antibodies targeting different epitopes

• Standard sample inclusion: Maintain reference samples across experiments for normalization

• Protocol repositories: Share detailed protocols through platforms like protocols.io

For PP2A research specifically, it's critical to acknowledge the limitations of phospho-specific antibodies and include appropriate controls for post-translational modifications that might affect antibody binding. Given the documented issues with phospho-Tyr307 antibodies, researchers should be particularly cautious when comparing results across studies that used different antibody clones .

How can I determine if previous research using PP2A antibodies remains valid in light of new specificity information?

Evaluating prior research requires careful analysis:

• Control evaluation: Assess whether appropriate controls were included (phosphatase treatments, mutants)

• Antibody identification: Determine which specific antibody clone was used

• Supporting evidence: Look for orthogonal methods that support the antibody-based findings

• Alternative explanations: Consider if findings could be explained by sensitivity to other modifications

• Replication attempts: Check if findings have been independently validated with different methods