PP2AA1 Antibody

What is PP2AA1 and what cellular functions does it regulate?

PP2AA1 (also referred to as PPP2CA) is the catalytic subunit alpha isoform of Protein Phosphatase 2A (PP2A), a major serine/threonine phosphatase that plays crucial roles in numerous cellular processes. PP2A functions as a heterotrimer consisting of a catalytic subunit (C), a structural subunit (A), and a variable regulatory subunit (B). The PP2AA1 catalytic subunit is involved in regulating:

• Multiple signaling pathways including Hippo, MAPK, and nuclear receptor pathways

• Cell cycle progression and G2/M checkpoint through WEE1 dephosphorylation

• Microtubule-associated protein (MAP) dynamics

• Oncogene regulation through dephosphorylation of proteins like MYC and FOXO3

• Cytoskeleton remodeling as part of STRIPAK complexes

• Inflammasome assembly via NLRP3 pyrin domain dephosphorylation

These diverse functions make PP2AA1 a critical target for investigation in various biological contexts, from normal cellular homeostasis to disease states.

How do I select the appropriate PP2AA1 antibody for my specific experimental application?

Antibody selection should be based on:

• Application compatibility: Different antibodies are optimized for specific applications such as Western blotting (WB), immunoprecipitation (IP), immunohistochemistry (IHC), or immunofluorescence (IF). For example, the PP2A-Aα/β Antibody (B-1) is validated for WB, IP, IF, and ELISA applications .

• Species reactivity: Verify that the antibody has been validated in your experimental species. The PP2A-alpha antibody from Abcam (ab137825) reacts with human and mouse samples , while the PP2A-Aα/β Antibody (B-1) can detect PP2A-Aα/β in mouse, rat, and human samples .

• Epitope recognition: Consider whether you need an antibody targeting a specific region or post-translational modification of PP2AA1. For example, ab137825 recognizes an epitope within amino acids 1-250 of human PPP2CA .

• Validation evidence: Review provided validation data including western blots showing predicted band sizes (35 kDa for PP2AA1) and examine citation records for evidence of successful application in published research.

• Clonality considerations: Monoclonal antibodies offer high specificity for single epitopes, while polyclonal antibodies may provide higher sensitivity by recognizing multiple epitopes.

What are the common experimental artifacts when working with PP2AA1 antibodies?

Several experimental artifacts can compromise research with PP2AA1 antibodies:

• Cross-reactivity with unmodified forms: Some antibodies marketed as specific for post-translationally modified forms (e.g., phospho-Tyr307 PP2A antibodies) have been shown to also detect unmodified PP2AA1 .

• Sensitivity to neighboring modifications: Antibody binding may be affected by modifications on residues near the target epitope. For example, the E155 and F-8 clones show reduced binding to PP2AA1 when Leu309 is methylated .

• Non-specific bands: Particularly in complex lysates, antibodies may bind to proteins of similar molecular weight to PP2AA1 (35 kDa).

• Batch-to-batch variation: Especially with polyclonal antibodies, different production lots may show variable specificity and sensitivity.

• Fixation artifacts: For immunohistochemistry and immunofluorescence applications, different fixation methods can affect epitope accessibility and antibody binding.

To minimize these artifacts, proper controls including knockdown/knockout samples are essential for validating antibody specificity in your experimental system.

How can I differentiate between PP2AA1 phosphorylation states using antibodies?

Distinguishing between PP2AA1 phosphorylation states requires careful consideration of antibody specificity:

Recent research has revealed significant limitations in antibodies previously believed to be phospho-specific. For example, extensive validation of commercially available phospho-Tyr307 PP2AC antibodies (including clones E155, F-8, and antibodies from R&D Systems) demonstrated that these antibodies cannot reliably differentiate between phosphorylated and unphosphorylated forms of PP2AA1 .

For accurate detection of phosphorylation states:

• Use alternative methods: Combine antibody-based detection with mass spectrometry to verify phosphorylation status.

• Implement appropriate controls: Include phosphatase treatment of samples to remove phosphorylation and compare signal intensity.

• Phospho-mimetic mutants: Generate Y307E or Y307D phospho-mimetic mutants and Y307F phospho-null mutants as controls for antibody validation.

• Consider neighboring modifications: Be aware that phosphorylation at Thr304 and methylation at Leu309 can affect antibody binding, potentially confounding interpretation of phosphorylation status at Tyr307 .

What are the most effective validation strategies for confirming PP2AA1 antibody specificity?

Comprehensive validation requires multiple approaches:

• Genetic knockout/knockdown controls:

  • siRNA/shRNA knockdown of PP2AA1

  • CRISPR/Cas9 knockout cell lines

  • Comparing signal in wild-type vs. knockout samples by Western blot

• Mutant expression systems:

  • Express tagged wild-type PP2AA1 alongside phospho-null mutants (e.g., Y307F)

  • Validate that antibodies marketed as phospho-specific do not detect phospho-null mutants

• Peptide competition assays:

  • Pre-incubate antibody with blocking peptides corresponding to the target epitope

  • Observe disappearance of specific signal

• Multiple antibody validation:

  • Use different antibodies recognizing distinct epitopes

  • Compare patterns of detection across applications

• Post-translational modification (PTM) manipulation:

  • Treat samples with phosphatases to remove phosphorylation

  • Expose cells to stimuli known to induce specific modifications (e.g., EGF treatment for tyrosine phosphorylation)

For example, researchers demonstrated that the E155 and F-8 antibodies detect both wild-type and Y307F mutant forms of PP2AA1 with equal intensity, revealing they cannot distinguish phosphorylated from unphosphorylated forms at Tyr307 .

How can I accurately assess PP2AA1 activity in complex biological samples?

Assessing PP2AA1 activity requires methods beyond simple antibody detection:

• PP2A activity assays:

  • Immunoprecipitate PP2A complexes using antibodies against the catalytic or structural subunits

  • Measure phosphatase activity using synthetic phosphopeptide substrates

  • Quantify released phosphate using malachite green or similar colorimetric methods

• Endogenous substrate phosphorylation:

  • Monitor phosphorylation status of well-characterized PP2A substrates

  • For example, measure RAF1 Ser259 phosphorylation, which is dephosphorylated by active PP2A

• Pharmacological approaches:

  • Use specific PP2A inhibitors (e.g., okadaic acid at appropriate concentrations)

  • Compare substrate phosphorylation with and without inhibitors

• Separation of PP2A complexes:

  • Employ immunoprecipitation to isolate specific PP2A holoenzymes

  • Assess activity of distinct PP2A complexes containing different regulatory subunits

In one study, researchers successfully measured PP2A activity in tolerized peritoneal macrophages, correlating increased PP2A activity with decreased TNF-α secretion upon second LPS stimulation , demonstrating the feasibility of activity measurements in complex cellular contexts.

What are the optimal protocols for using PP2AA1 antibodies in immunofluorescence applications?

For successful immunofluorescence with PP2AA1 antibodies:

• Fixation optimization:

  • Methanol fixation has been successfully used for PP2AA1 detection in HeLa cells

  • Test both paraformaldehyde (4%, 10-15 minutes) and methanol (100%, 10 minutes at -20°C) fixation methods

  • Some epitopes may be masked by certain fixation methods

• Antibody dilution and incubation:

  • Start with manufacturer-recommended dilutions (e.g., 1/500 for ab137825)

  • For primary antibody, incubate overnight at 4°C or 1-2 hours at room temperature

  • For secondary antibody, typical incubations are 1 hour at room temperature

• Controls and counterstaining:

  • Include negative controls (primary antibody omission)

  • Consider dual staining with established markers (e.g., alpha-tubulin as demonstrated with ab137825)

  • Use DAPI for nuclear counterstaining

• Signal amplification and detection:

  • For weak signals, consider tyramide signal amplification

  • Use confocal microscopy for optimal subcellular localization

• Image acquisition settings:

  • Adjust exposure to avoid signal saturation

  • Maintain consistent settings between experimental and control samples

For example, successful visualization of PP2AA1 in HeLa cells has been achieved using methanol fixation with ab137825 at 1/500 dilution, counterstained with alpha-tubulin .

How can I optimize Western blot protocols for consistent PP2AA1 detection?

Optimization strategies for Western blotting include:

• Sample preparation:

  • Use phosphatase inhibitors in lysis buffers to preserve phosphorylation status

  • For total PP2AA1, standard RIPA or NP-40 buffers are suitable

  • Load appropriate protein amounts (30-50 μg total protein is typical)

• Gel selection and transfer conditions:

  • 10-12% SDS-PAGE gels are appropriate for resolving the 35 kDa PP2AA1

  • Semi-dry or wet transfer systems work well (20-30V overnight or 100V for 1 hour)

  • Use PVDF membranes for optimal protein binding

• Blocking and antibody incubation:

  • 5% non-fat dry milk or BSA in TBST for blocking (1 hour at room temperature)

  • For phospho-specific detection, BSA is preferred over milk

  • Typical primary antibody dilutions: 1/1000 for ab137825

  • Incubate primary antibody overnight at 4°C

• Detection and quantification:

  • Use HRP-conjugated secondary antibodies with ECL detection

  • For quantification, ensure exposure times avoid signal saturation

  • Normalize to appropriate loading controls (e.g., GAPDH, β-actin)

• Stripping and reprobing:

  • Gentle stripping buffers can allow sequential probing for phosphorylated and total protein

  • Validate complete stripping before reprobing

The expected band size for PP2AA1 is 35 kDa, and this should be verified using appropriate molecular weight markers .

What controls should be implemented when using PP2AA1 antibodies for immunoprecipitation?

Essential controls for immunoprecipitation experiments include:

• Input control:

  • Always run an aliquot of starting material (5-10% of IP amount)

  • Use for comparison to immunoprecipitated material

• Negative antibody control:

  • Perform parallel IP with isotype-matched irrelevant antibody

  • For mouse monoclonal antibodies like PP2A-Aα/β Antibody (B-1), use mouse IgG2a

• Beads-only control:

  • Incubate lysate with capture matrix without antibody

  • Identifies proteins binding non-specifically to beads

• Competitive blocking:

  • Where available, use immunizing peptide to block antibody binding

  • Santa Cruz offers a neutralizing peptide for PP2A-Aα/β (B-1) antibody

• Reciprocal IP:

  • For protein-protein interaction studies, perform IP with antibodies against each partner

  • Confirms interactions from both perspectives

• Validation in knockdown/knockout samples:

  • Perform IP in cells where PP2AA1 expression is reduced/eliminated

  • Demonstrates specificity of immunoprecipitation

For PP2AA1 complex studies, consider using alternative antibodies against different PP2A subunits (A, B, or C) to validate interactions and complex formation.

How should I interpret conflicting results between different PP2AA1 antibodies?

When faced with conflicting results:

• Epitope differences:

  • Different antibodies may recognize distinct regions of PP2AA1

  • Post-translational modifications may differentially affect epitope accessibility

  • For example, the E155 antibody's ability to detect PP2AA1 is significantly reduced when Leu309 is methylated

• Clonality considerations:

  • Monoclonal antibodies target single epitopes, while polyclonal antibodies recognize multiple epitopes

  • Polyclonal antibodies may maintain signal despite modification of individual epitopes

• Sensitivity to neighboring modifications:

  • The F-8 clone binds less efficiently to peptides phosphorylated at Thr304

  • All three antibodies studied (E155, F-8, R&D) bind methylated Leu309 forms with reduced efficiency

• Validation approach:

  • Create a validation matrix with multiple antibodies across different applications

  • Include knockout/knockdown controls for each antibody

  • Use orthogonal methods (mass spectrometry) to confirm antibody results

• Resolution strategies:

  • When antibodies disagree, trust results from antibodies with most extensive validation

  • For critical findings, confirm with non-antibody-based methods

  • Consider that both results may be correct if detecting different subpopulations of PP2AA1

The study by Frohner et al. (2020) highlights how reinterpretation of previous work may be necessary when antibody specificity is thoroughly examined .

What are the implications of recent findings regarding phospho-Tyr307 PP2AA1 antibody specificity for previous research?

The discovery that widely used phospho-Tyr307 antibodies cannot differentiate between phosphorylated and unphosphorylated forms has profound implications:

This case highlights the critical importance of rigorous antibody validation and demonstrates how insufficient validation can lead an entire field in potentially misleading directions .

What experimental design would definitively resolve PP2AA1 phosphorylation status in biological samples?

A comprehensive experimental design would include:

• Multiple detection approaches:

  • Antibody-based detection with extensively validated antibodies

  • Mass spectrometry analysis of immunoprecipitated PP2AA1

  • Phospho-protein staining (Pro-Q Diamond) followed by total protein staining

• Genetic manipulation:

  • Expression of tagged wild-type PP2AA1 alongside phospho-null (Y307F) and phospho-mimetic (Y307E) mutants

  • CRISPR-mediated genomic editing to create endogenous Y307F mutations

• Pharmacological interventions:

  • Treatment with phosphatase inhibitors (okadaic acid, calyculin A)

  • Tyrosine kinase inhibitors targeting kinases implicated in Y307 phosphorylation

  • Phosphatase treatment of cellular extracts

• Functional correlation:

  • Parallel measurement of PP2A activity using phosphatase assays

  • Correlation of activity with phosphorylation status

  • Assessment of downstream substrate phosphorylation

• Temporal dynamics:

  • Time-course experiments following stimulation

  • Correlation of changes in phosphorylation with functional outcomes

This multi-faceted approach would minimize reliance on any single detection method and provide multiple lines of evidence regarding PP2AA1 phosphorylation status and its functional significance.

How can advanced proteomics approaches enhance PP2AA1 antibody validation and phosphorylation site mapping?

Advanced proteomics offers powerful solutions for PP2AA1 research:

• Parallel Reaction Monitoring (PRM):

  • Targeted mass spectrometry approach for precise quantification of specific PP2AA1 phosphopeptides

  • Can distinguish between different phosphorylated forms with site-specific resolution

  • Provides absolute quantification of modification stoichiometry

• Crosslinking Mass Spectrometry (XL-MS):

  • Maps interaction interfaces between PP2AA1 and regulatory partners

  • Identifies conformational changes induced by phosphorylation

  • Provides structural insights complementary to antibody detection

• Phosphoproteomics workflow integration:

  • Enrichment of phosphopeptides using TiO2 or immobilized metal affinity chromatography

  • High-resolution LC-MS/MS analysis

  • Parallel antibody validation with the same samples

• Thermal Proteome Profiling (TPP):

  • Measures changes in protein thermal stability upon modification

  • Can detect functional consequences of PP2AA1 phosphorylation

  • Provides proteome-wide context for PP2AA1 regulation

These approaches provide antibody-independent confirmation of PP2AA1 phosphorylation status and offer deeper insights into the functional consequences of these modifications.

What roles do PP2AA1 antibodies play in investigating PP2A complexes and STRIPAK assemblies?

PP2AA1 antibodies are invaluable tools for studying complex PP2A assemblies:

• PP2A holoenzyme composition analysis:

  • Immunoprecipitation with PP2AA1 antibodies followed by mass spectrometry

  • Identification of regulatory B subunits associated with specific complexes

  • Quantification of complex stoichiometry in different cellular contexts

• STRIPAK complex investigation:

  • PP2AA1 is part of striatin-interacting phosphatase and kinase (STRIPAK) complexes that regulate multiple signaling pathways

  • Antibodies enable purification of intact STRIPAK complexes

  • Co-immunoprecipitation experiments reveal STRIPAK component interactions

• Dynamic complex assembly studies:

  • Time-course immunoprecipitation following stimulus

  • Tracking changes in complex composition during cellular responses

  • Correlation with functional outcomes

• Spatial organization analysis:

  • Immunofluorescence with PP2AA1 antibodies reveals subcellular localization

  • Co-localization studies with other STRIPAK components

  • Super-resolution microscopy for nanoscale organization

• Functional studies of assembled complexes:

  • Activity assays of immunopurified complexes

  • Reconstitution experiments with purified components

  • Structure-function analysis of mutant complexes

These approaches contribute to understanding how PP2AA1 functions within larger molecular assemblies to regulate diverse cellular processes including Hippo, MAPK, and cytoskeleton remodeling pathways .

What are the most critical considerations for researchers planning to use PP2AA1 antibodies in their studies?

Researchers should prioritize:

• Rigorous validation:

  • Never rely solely on manufacturer claims about antibody specificity

  • Implement genetic controls (knockdown/knockout)

  • Use multiple antibodies targeting different epitopes

  • Be especially cautious with antibodies targeting post-translational modifications

• Application-specific optimization:

  • Validate each antibody for specific applications (WB, IP, IF, IHC)

  • Optimize conditions including fixation, blocking, and antibody concentration

  • Consider that an antibody working well in one application may not work in others

• Literature awareness:

  • Stay informed about antibody validation literature

  • Recognize that prior findings may require reinterpretation if based on inadequately validated antibodies

  • The case of phospho-Tyr307 antibodies demonstrates how widely used reagents may have unexpected properties

• Complementary approaches:

  • Supplement antibody-based detection with orthogonal methods

  • Consider mass spectrometry for definitive determination of post-translational modifications

  • Use functional assays to correlate antibody signals with biological activity

• Detailed reporting:

  • Document all validation steps in publications

  • Report catalog numbers, clone information, and experimental conditions

  • Share validation data to benefit the research community

These considerations ensure robust, reproducible research with PP2AA1 antibodies and contribute to addressing the broader "reproducibility crisis" in biomedical research .

How might emerging antibody technologies address current limitations in PP2AA1 research?

Innovative approaches hold promise for overcoming current challenges:

• Recombinant antibody technology:

  • Generation of recombinant antibodies with precisely defined binding characteristics

  • Elimination of batch-to-batch variation through recombinant production

  • Engineering of antibodies with enhanced specificity for post-translational modifications

• Single-domain antibodies (nanobodies):

  • Smaller size enables access to epitopes inaccessible to conventional antibodies

  • Potential for improved specificity for phosphorylated residues

  • Applications in live-cell imaging of dynamic PP2AA1 regulation

• Proximity labeling approaches:

  • Antibody-enzyme fusions for proximity-dependent labeling

  • Mapping of PP2AA1 interaction networks in living cells

  • Temporal resolution of dynamic complex formation

• Intrabodies and biosensors:

  • Expression of engineered antibody fragments in living cells

  • Real-time monitoring of PP2AA1 modifications and interactions

  • Correlation with functional outcomes in intact systems

• Synthetic binding proteins:

  • Designed proteins with high specificity for particular PP2AA1 forms

  • Rational engineering based on structural information

  • Potential for absolute specificity for phosphorylated versus unphosphorylated forms