PPP2R2A Antibody

What is PPP2R2A and why is it important in cellular functions?

PPP2R2A, also known as Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B alpha isoform (PP2A subunit B isoform B55-alpha), functions as a regulatory component of the PP2A holoenzyme complex. The B regulatory subunit modulates substrate selectivity and catalytic activity while also directing the localization of the catalytic enzyme to specific subcellular compartments. PPP2R2A is essential for serine/threonine-protein phosphatase 2A-mediated dephosphorylation of WEE1, preventing its ubiquitin-mediated proteolysis, increasing WEE1 protein levels, and promoting the G2/M checkpoint . This protein plays a crucial role in maintaining genomic stability through its involvement in DNA damage response pathways, making it an important research target in cancer biology and cell cycle regulation studies.

What applications are PPP2R2A antibodies suitable for?

PPP2R2A antibodies are suitable for multiple experimental applications including Western Blotting (WB), Immunofluorescence (IF), and Immunohistochemistry on paraffin-embedded sections (IHC-P) . When selecting an antibody for your research, consider the specific requirements of your experimental design. For protein detection and quantification in cell or tissue lysates, Western Blotting is most appropriate. For visualizing subcellular localization patterns, Immunofluorescence provides better spatial resolution. For examining protein expression in the context of tissue architecture, Immunohistochemistry offers valuable insights. Some PPP2R2A antibodies may also be suitable for ELISA techniques depending on the specific product .

How do I select the appropriate PPP2R2A antibody for my specific research question?

When selecting a PPP2R2A antibody, consider these methodological factors:

• Target epitope location: Different antibodies target various regions of PPP2R2A (e.g., N-terminal AA 43-71, full-length AA 1-447, or other specific domains) . The epitope location can affect antibody performance in different applications, especially if your research focuses on specific protein domains or if post-translational modifications might mask certain epitopes.

• Species reactivity: Verify the antibody's reactivity with your experimental model. Some PPP2R2A antibodies react with human samples only, while others cross-react with mouse, rat, and other species .

• Clonality: Most available PPP2R2A antibodies are polyclonal (derived from multiple B-cell lineages), which provides broader epitope recognition but potentially higher batch-to-batch variability compared to monoclonal antibodies .

• Application-specific validation: Ensure the antibody has been validated for your specific application with supporting data from the manufacturer or published literature.

How does PPP2R2A regulate DNA damage response pathways?

PPP2R2A plays a critical role in DNA damage response through its regulation of ATM (Ataxia Telangiectasia Mutated) kinase activity. PPP2R2A-containing complexes directly dephosphorylate ATM at specific regulatory sites including S367, S1893, and S1981, which controls ATM retention at double-strand break (DSB) sites . When PPP2R2A function is attenuated, increased ATM phosphorylation dramatically upregulates the activity of the downstream effector kinase CHK2, resulting in G1 to S-phase cell cycle arrest and downregulation of BRCA1 and RAD51 .

This regulatory mechanism has significant implications for DNA repair capacity, as the PPP2R2A-ATM axis controls the high-fidelity homologous recombination (HR) repair pathway. Experimental evidence demonstrates that blocking PPP2R2A impairs HR repair efficiency, potentially creating therapeutic vulnerabilities in cancer cells with altered PPP2R2A expression . This makes PPP2R2A status a potential biomarker for predicting sensitivity to targeted therapies like PARP inhibitors that exploit deficiencies in DNA repair pathways.

What methodological considerations should be taken when using PPP2R2A antibodies in DNA repair studies?

When using PPP2R2A antibodies in DNA repair studies, researchers should implement these methodological approaches:

• Combine antibody-based detection with functional assays: While antibody detection of PPP2R2A provides information about protein levels and localization, functional phosphatase assays should be employed to correlate protein expression with enzymatic activity.

• Consider post-translational modifications: PPP2R2A function is regulated by various post-translational modifications. Use phospho-specific antibodies when investigating PPP2R2A's role in ATM dephosphorylation at specific sites (S367, S1893, S1981) .

• Include appropriate controls: When studying PPP2R2A in DNA repair contexts, include positive controls (cells with known PPP2R2A expression) and negative controls (PPP2R2A knockdown or knockout systems) to validate antibody specificity.

• Validate findings with orthogonal methods: Complement antibody-based detection with gene expression analysis, phosphatase activity assays, and functional DNA repair readouts (e.g., HR reporter assays, RAD51 foci formation).

How can PPP2R2A antibodies be used to investigate potential biomarkers in cancer research?

PPP2R2A has been identified as commonly downregulated in non-small cell lung carcinomas, suggesting its status may serve as a predictive biomarker for PARP inhibitor response . When using PPP2R2A antibodies for biomarker investigations:

• Standardize tissue processing and staining protocols: Consistent fixation, antigen retrieval, and staining procedures are essential for quantitative comparisons across patient samples.

• Employ quantitative image analysis: Use digital pathology platforms with standardized scoring algorithms to objectively quantify PPP2R2A expression levels rather than relying solely on qualitative assessments.

• Correlate with clinical outcomes: Integrate PPP2R2A expression data with patient treatment responses, particularly to therapies targeting DNA repair pathways like PARP inhibitors.

• Validate across multiple cohorts: Confirm biomarker findings in independent patient cohorts and using different antibody clones to ensure robustness of the association.

What are common pitfalls in Western blot experiments using PPP2R2A antibodies and how can they be addressed?

Common challenges when using PPP2R2A antibodies for Western blotting include:

• Multiple banding patterns: PPP2R2A has a predicted molecular weight of approximately 55 kDa, but post-translational modifications or splice variants may result in additional bands. To address this:

  • Include positive and negative controls to confirm band identity

  • Use genetic approaches (siRNA/shRNA) to validate specific bands

  • Consider the epitope location of your antibody, as N-terminal or C-terminal targeting may affect banding patterns

• Cross-reactivity with other PP2A regulatory subunits: The PP2A family includes multiple regulatory subunits with sequence homology. To minimize cross-reactivity:

  • Select antibodies validated against a panel of PP2A family members

  • Use appropriate blocking conditions to reduce non-specific binding

  • Consider antibodies targeting unique regions of PPP2R2A rather than conserved domains

• Variable expression levels across cell types: PPP2R2A expression varies significantly between tissues and cell lines. To optimize detection:

  • Adjust protein loading amounts based on expected expression levels

  • Optimize exposure times to detect low abundance signals without saturating stronger signals

  • Consider enrichment strategies (e.g., immunoprecipitation) for low-expressing samples

How can researchers optimize immunofluorescence protocols for PPP2R2A detection?

To optimize immunofluorescence protocols for detecting PPP2R2A:

• Fixation method selection: Different fixation protocols can affect epitope accessibility.

  • Paraformaldehyde (4%) works well for preserving protein localization

  • Methanol fixation may better expose certain epitopes but can disrupt membrane structures

  • Test multiple fixation protocols if initial attempts yield poor signal

• Antigen retrieval considerations:

  • For formalin-fixed tissues, heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) often improves PPP2R2A detection

  • For cultured cells, mild detergent permeabilization (0.1-0.3% Triton X-100) generally provides sufficient access to intracellular epitopes

• Signal amplification strategies:

  • Tyramide signal amplification can enhance detection of low-abundance targets

  • Secondary antibody selection (highly cross-adsorbed versions) minimizes background

  • Extended primary antibody incubation (overnight at 4°C) often improves signal-to-noise ratio

• Multiplexing considerations:

  • When co-staining PPP2R2A with phospho-proteins (e.g., phospho-ATM), sequential rather than simultaneous antibody incubation may yield better results

  • Choose fluorophores with minimal spectral overlap when examining colocalization with DNA repair factors

How should researchers interpret changes in PPP2R2A expression in relation to DNA repair capacity?

When interpreting PPP2R2A expression changes in the context of DNA repair:

• Correlation with ATM phosphorylation status: Decreased PPP2R2A expression typically correlates with increased ATM phosphorylation at S367, S1893, and S1981 sites . This relationship should be experimentally validated in your specific experimental system.

• Downstream effects on HR repair proteins: Loss of PPP2R2A function leads to downregulation of BRCA1 and RAD51, key mediators of homologous recombination repair . When observing PPP2R2A expression changes, researchers should examine these downstream effectors to confirm functional impact on the HR pathway.

• Cell cycle considerations: PPP2R2A regulates the G2/M checkpoint through its impact on WEE1 stability . Changes in PPP2R2A expression may therefore have cell cycle-specific effects that could confound interpretation of DNA repair phenotypes if not properly controlled for.

• Context-dependent function: PPP2R2A function may be cell type-specific or influenced by the genetic background of the experimental system. Comparative analysis across multiple cell lines or primary tissues can help establish the generalizability of observed phenotypes.

What strategies can be employed to validate PPP2R2A antibody specificity for research applications?

To ensure PPP2R2A antibody specificity:

• Genetic validation approaches:

  • Use siRNA/shRNA knockdown or CRISPR/Cas9 knockout of PPP2R2A to confirm signal reduction

  • Rescue experiments with exogenous PPP2R2A expression in knockout systems can further confirm specificity

  • If available, test the antibody in tissues from PPP2R2A knockout animal models

• Peptide competition assays:

  • Pre-incubation of the antibody with the immunizing peptide should abolish specific signals

  • Include a non-competing peptide control to confirm specificity of the competition

• Cross-validation with multiple antibodies:

  • Compare results using antibodies targeting different epitopes of PPP2R2A

  • Concordant results across antibodies increase confidence in specificity

• Orthogonal detection methods:

  • Correlate antibody-based detection with mRNA expression analysis

  • Mass spectrometry validation of immunoprecipitated proteins can confirm antibody target identity

How can PPP2R2A antibodies be utilized in studies investigating cancer therapeutic response?

PPP2R2A antibodies can be strategically employed in cancer therapeutic studies through:

• Biomarker stratification: PPP2R2A downregulation in tumors may predict sensitivity to PARP inhibitors . Researchers can use immunohistochemistry with validated PPP2R2A antibodies to stratify patient-derived xenograft models or clinical samples for therapeutic testing.

• Mechanism-of-action studies: Combining PPP2R2A expression analysis with functional assays of DNA repair capacity can elucidate the mechanism by which altered PPP2R2A levels influence therapeutic responses.

• Combination therapy investigations: PPP2R2A antibodies can help monitor changes in expression or localization following treatment with various therapeutic agents, potentially identifying synergistic combinations that target PPP2R2A-regulated pathways.

• Resistance mechanism identification: Comparing PPP2R2A expression and phosphatase activity in treatment-naive versus resistant tumor samples may reveal adaptive mechanisms that restore DNA repair capacity despite initial PPP2R2A deficiency.

What emerging applications of PPP2R2A antibodies should researchers be aware of?

Emerging applications for PPP2R2A antibodies include:

• Single-cell analysis: Adapting PPP2R2A antibodies for mass cytometry (CyTOF) or imaging mass cytometry enables characterization of PPP2R2A expression heterogeneity at the single-cell level within complex tissues.

• Proximity ligation assays: These techniques can reveal protein-protein interactions between PPP2R2A and its binding partners (such as ATM) with spatial resolution in intact cells, providing insights into context-dependent complex formation.

• Live-cell imaging applications: Development of conformation-specific antibodies or antibody fragments compatible with live-cell imaging could enable real-time monitoring of PPP2R2A activity during DNA damage responses.

• Therapeutic development: PPP2R2A antibodies can facilitate screening of small molecules designed to modulate PP2A function, potentially identifying compounds that restore PPP2R2A activity in cancers where it is downregulated.