PP2AA2 Antibody

What is PP2A-alpha and what cellular functions does it regulate?

PP2A-alpha is the catalytic subunit of Protein Phosphatase 2A, a major serine/threonine phosphatase in eukaryotic cells. The PP2A holoenzyme typically exists as a trimeric complex consisting of a catalytic subunit, a regulatory subunit, and a variable subunit. PP2A-alpha plays crucial roles in numerous cellular processes by dephosphorylating target proteins. It serves as the major phosphatase for microtubule-associated proteins (MAPs) and modulates the activity of several kinases including phosphorylase B kinase, casein kinase 2, mitogen-stimulated S6 kinase, and MAP-2 kinase. Through these interactions, PP2A regulates cell division, homeostasis, and apoptosis in various cell types and organisms from yeast to humans . The enzyme is involved in protecting centromeric cohesin during meiosis and participates in critical developmental processes through its ability to dephosphorylate key signaling molecules like RAF1, WEE1, MYC, and FOXO3 .

What are the recommended applications for PP2A-alpha antibodies?

PP2A-alpha antibodies are versatile tools that can be employed across multiple experimental applications. Based on validated research protocols, these antibodies are suitable for several techniques including Western blotting (WB), immunohistochemistry on paraffin-embedded samples (IHC-P), and immunocytochemistry/immunofluorescence (ICC/IF). For Western blotting, PP2A antibodies provide reliable detection of both alpha and beta isoforms, allowing researchers to analyze protein expression levels and post-translational modifications in cell and tissue lysates . In immunohistochemistry applications, these antibodies enable visualization of PP2A localization in tissue sections, particularly useful for brain tissue where PP2A plays significant roles in neuronal signaling . For immunofluorescence, the antibodies permit detailed subcellular localization studies to examine PP2A distribution in fixed cells, offering insights into its spatial regulation and potential interactions with other cellular components .

Which species reactivity should be considered when selecting a PP2A-alpha antibody?

When selecting a PP2A-alpha antibody, researchers should carefully consider species cross-reactivity to ensure compatibility with their experimental models. Available commercial antibodies demonstrate varied cross-reactivity profiles. Some antibodies, like the PP2A Monoclonal Antibody (7A6), recognize the C-terminal region of both PP2A alpha and beta isoforms in multiple species including human, mouse, rat, and Saccharomyces cerevisiae samples . Other antibodies, such as the rabbit polyclonal anti-PP2A-alpha antibody, react specifically with human, mouse, and rat samples . The high degree of evolutionary conservation in the PP2A catalytic subunit sequence explains this broad cross-reactivity across species. Researchers should verify the validated species reactivity in product documentation and consider preliminary testing in their specific experimental system, especially when working with less common model organisms not typically tested by manufacturers .

What controls should be included when using PP2A-alpha antibodies?

Proper experimental controls are essential for validating results obtained with PP2A-alpha antibodies. At minimum, researchers should include a negative control (omitting primary antibody) to assess non-specific binding of the secondary detection system. For Western blotting, a positive control consisting of lysate from cells known to express PP2A (such as HeLa or HEK293 cells) should be included alongside experimental samples . When available, knockout cell lines provide the gold standard for antibody validation, as demonstrated in recent characterization studies of regulatory subunit antibodies . For immunoprecipitation experiments, researchers should include an isotype control antibody to identify non-specific protein interactions. When using multiple detection methods (e.g., WB and IHC), concordance between techniques strengthens the validity of findings. Additionally, peptide competition assays, where the antibody is pre-incubated with the immunizing peptide, can confirm binding specificity by demonstrating signal reduction or elimination in the presence of the competing antigen .

How can researchers distinguish between PP2A-alpha and PP2A-beta isoforms in their experiments?

Distinguishing between PP2A-alpha (PPP2CA) and PP2A-beta (PPP2CB) isoforms presents a significant challenge due to their high sequence homology. Some antibodies, like the PP2A Monoclonal Antibody (7A6), recognize both alpha and beta isoforms by targeting the conserved C-terminal region . For isoform-specific detection, researchers should select antibodies raised against unique epitopes, typically within the amino acid regions 1-50 or 200-300, where sequence divergence exists between isoforms . Combining immunological techniques with molecular approaches can enhance specificity. For instance, researchers can use siRNA-mediated knockdown of individual isoforms followed by Western blotting to confirm antibody specificity. Alternatively, recombinant expression of tagged isoforms can serve as positive controls for antibody validation. Mass spectrometry-based proteomics following immunoprecipitation offers another approach for definitive isoform identification, allowing researchers to detect unique peptides that differentiate between the alpha and beta catalytic subunits. These complementary approaches ensure accurate isoform identification, critical for understanding their potentially distinct functions in different cellular contexts .

What are the optimal protocols for detecting PP2A-alpha in brain tissue samples?

Detecting PP2A-alpha in brain tissue requires specialized protocols due to the complex nature of neural tissue. For immunohistochemistry on paraffin-embedded brain sections, researchers should optimize antigen retrieval methods, with heat-induced epitope retrieval in citrate buffer (pH 6.0) generally providing good results. Blocking with 5-10% normal serum from the same species as the secondary antibody for at least 1 hour minimizes background staining, particularly important in brain tissue with its diverse cell populations . For Western blotting of brain lysates, tissue should be rapidly collected and flash-frozen to preserve phosphorylation states. Homogenization in RIPA buffer supplemented with phosphatase inhibitors (including sodium fluoride, sodium orthovanadate, and β-glycerophosphate) protects against ex vivo dephosphorylation events that could alter results. Since PP2A regulatory subunits like PPP2R5D are abundantly expressed in the brain and linked to neurodevelopmental disorders, researchers should consider dual-staining approaches to investigate catalytic and regulatory subunit co-localization patterns . For immunofluorescence on cultured neurons, fixation with 4% paraformaldehyde for 15 minutes at room temperature followed by gentle permeabilization with 0.1% Triton X-100 preserves subcellular structures while enabling antibody access to intracellular targets .

What strategies can address potential cross-reactivity issues when using PP2A-alpha antibodies?

Cross-reactivity remains a significant challenge when working with phosphatase antibodies due to structural similarities between phosphatase families. To address this issue, researchers should implement multiple validation strategies. Independent validation using different antibodies targeting distinct epitopes of PP2A-alpha provides confirmation through concordance of results . For definitive validation, genetic approaches using CRISPR/Cas9-mediated knockout cell lines paired with corresponding parental controls offer the gold standard for demonstrating antibody specificity, as highlighted in recent standardized antibody characterization protocols . When knockout models are unavailable, siRNA or shRNA-mediated knockdown provides an alternative approach, with the degree of signal reduction correlating with knockdown efficiency. Peptide competition assays, where pre-incubation of the antibody with immunizing peptide blocks specific binding, offer another validation method. For applications involving complex samples like brain tissue, researchers should consider dual-labeling with independently validated markers to confirm PP2A-alpha localization patterns. Finally, mass spectrometry analysis following immunoprecipitation can definitively identify all proteins pulled down by the antibody, revealing potential cross-reactive species and confirming target enrichment .

How can researchers investigate PP2A-alpha interactions with STRIPAK complexes in experimental systems?

Investigating PP2A-alpha interactions with Striatin-Interacting Phosphatase And Kinase (STRIPAK) complexes requires specialized approaches that preserve protein-protein interactions. Researchers should begin with co-immunoprecipitation assays using PP2A-alpha antibodies under gentle lysis conditions (such as 0.3% CHAPS or NP-40 detergent) that maintain complex integrity. Sequential immunoprecipitation, where complexes are eluted from one antibody and re-precipitated with another, can identify specific STRIPAK sub-complexes containing PP2A-alpha . For spatial analysis, proximity ligation assays (PLA) offer in situ visualization of PP2A-alpha interactions with STRIPAK components like striatin, with positive signals indicating proteins are within 40nm of each other. To map dynamic interactions, researchers can employ BioID or APEX2 proximity labeling, where a biotin ligase fused to PP2A-alpha biotinylates nearby proteins, allowing identification of transient interactions that might be missed by co-immunoprecipitation. For functional studies, researchers should systematically manipulate expression of STRIPAK components and assess effects on PP2A-alpha localization and substrate dephosphorylation, particularly within signaling pathways like Hippo, MAPK, and cytoskeleton remodeling known to be regulated by STRIPAK complexes . Mass spectrometry analysis following affinity purification can comprehensively identify all STRIPAK components associated with PP2A-alpha under different cellular conditions, providing insights into context-dependent complex formation and regulation .

What are the critical steps for successful Western blotting with PP2A-alpha antibodies?

Successful Western blotting with PP2A-alpha antibodies requires careful attention to several critical steps. Sample preparation is paramount—cells or tissues should be lysed in buffer containing phosphatase inhibitors (10mM sodium fluoride, 1mM sodium orthovanadate) to preserve the phosphorylation state of PP2A and its substrates. Protein loading should be standardized (typically 20-30μg per lane) and verified with housekeeping protein controls like GAPDH or β-actin . During electrophoresis, 10-12% polyacrylamide gels provide optimal resolution for PP2A-alpha, which has a molecular weight of approximately 36 kDa. Transfer efficiency to nitrocellulose or PVDF membranes should be confirmed using Ponceau S staining before proceeding to immunodetection. For blocking, 5% non-fat dry milk in TBST is generally effective, though some antibodies may perform better with 3-5% BSA blocking solution, particularly when analyzing phosphorylation states . Primary antibody incubation should occur at 4°C overnight using manufacturer-recommended dilutions (typically 1:1000 to 1:2000). After washing, HRP-conjugated secondary antibodies at 1:5000 to 1:10000 dilutions provide good signal-to-noise ratios. For detection, enhanced chemiluminescence substrates are suitable, with exposure times optimized to avoid signal saturation that could compromise quantitative analysis. When comparing expression levels across samples, researchers must ensure linear range detection and normalize to loading controls for accurate quantification .

What troubleshooting approaches can researchers apply when PP2A-alpha antibodies show weak or non-specific signals?

When encountering weak or non-specific signals with PP2A-alpha antibodies, researchers should implement a systematic troubleshooting approach. For weak signals, first verify protein loading by analyzing housekeeping proteins or total protein stains like Ponceau S or Coomassie. Increasing primary antibody concentration or extending incubation time (up to 48 hours at 4°C) may enhance detection sensitivity, as may switching to more sensitive chemiluminescent substrates or using signal amplification systems . Non-specific bands require different interventions—researchers should optimize blocking conditions by testing different blocking agents (milk versus BSA) and concentrations (3-5%), potentially adding 0.1-0.3% Tween-20 to reduce background. Increasing washing duration and number of washes (5-6 washes of 10 minutes each) can significantly reduce non-specific binding. If multiple bands persist, researchers should consider the possibility of PP2A post-translational modifications, proteolytic fragments, or cross-reactivity with related phosphatases. Comparing results with different antibodies targeting distinct epitopes can help identify truly specific signals. For applications in new cell types or species, researchers should validate the antibody using positive controls (cells known to express PP2A) and negative controls (PP2A-depleted samples through siRNA/shRNA knockdown or CRISPR knockout when available) . Finally, peptide competition assays can confirm signal specificity by demonstrating signal elimination when the antibody is pre-incubated with the immunizing peptide .

How should researchers interpret complex patterns of PP2A-alpha localization in immunofluorescence experiments?

Interpreting complex patterns of PP2A-alpha localization in immunofluorescence experiments requires careful analysis and appropriate controls. PP2A-alpha typically shows both cytoplasmic and nuclear distribution, with potential enrichment in specific subcellular compartments depending on cell type and physiological state. Researchers should first validate antibody specificity using siRNA knockdown or CRISPR knockout controls to confirm that the observed pattern is genuine . To dissect complex localization patterns, co-staining with organelle markers provides crucial context—markers for the nucleus (DAPI), endoplasmic reticulum (calreticulin), Golgi apparatus (GM130), mitochondria (MitoTracker), and cytoskeleton (phalloidin for F-actin, α-tubulin for microtubules) help identify specific compartments where PP2A-alpha may be enriched. Super-resolution microscopy techniques like structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy offer enhanced resolution for detailed localization studies . Cell cycle-dependent changes in PP2A-alpha distribution should be considered, as phosphatase localization often shifts during mitosis. Researchers should examine cells at different cell cycle stages, identifiable through nuclear morphology or specific markers. Treatment with phosphatase inhibitors (okadaic acid or calyculin A) or kinase inhibitors may reveal dynamic aspects of PP2A-alpha localization dependent on phosphorylation-dephosphorylation cycles. Finally, co-staining with regulatory subunits or known substrates can provide functional context to the observed localization patterns, linking spatial distribution to potential biological activities .

What considerations are important when designing PP2A-alpha knockdown or knockout validation experiments?

Designing rigorous PP2A-alpha knockdown or knockout validation experiments requires careful consideration of several factors. When planning siRNA or shRNA knockdown experiments, researchers should design multiple targeting sequences to control for off-target effects, with at least two independent sequences showing similar phenotypes providing stronger evidence for specificity. Quantification of knockdown efficiency through both qRT-PCR (mRNA level) and Western blotting (protein level) is essential, with 70-90% reduction typically needed to observe functional consequences . For CRISPR/Cas9-mediated knockout approaches, researchers should design multiple guide RNAs targeting early exons and confirm gene disruption through sequencing. Complete knockout of PP2A-alpha may be lethal in some cell types due to its essential functions, potentially necessitating conditional knockout systems like Cre-loxP or inducible CRISPR approaches . Rescue experiments provide the gold standard for specificity validation—reintroduction of PP2A-alpha cDNA (preferably with silent mutations rendering it resistant to the knockdown construct) should reverse the observed phenotypes. When analyzing phenotypes, researchers should consider the potential compensatory upregulation of the PP2A-beta isoform or other phosphatases, which might mask the full impact of PP2A-alpha loss. Phosphorylation levels of known PP2A substrates serve as functional readouts of reduced phosphatase activity. Finally, researchers should assess long-term versus acute depletion effects, as adaptive responses may emerge over time that were not apparent in short-term knockdown experiments .

How can researchers effectively use PP2A-alpha antibodies to study its role in neurodevelopmental disorders?

Investigating PP2A-alpha's role in neurodevelopmental disorders requires specialized approaches tailored to neuronal systems. Researchers should begin with immunohistochemical analysis of post-mortem brain tissue from patients with relevant disorders compared to age-matched controls, focusing on regions implicated in the specific condition (e.g., prefrontal cortex in autism spectrum disorders) . For mechanistic studies, iPSC-derived neurons from patients or CRISPR-engineered lines carrying disease-associated mutations provide valuable model systems. When analyzing these models, researchers should examine both PP2A-alpha expression levels and its association with regulatory subunits like PPP2R5D, mutations in which are linked to intellectual disability and autism spectrum disorders . Co-immunoprecipitation followed by mass spectrometry can identify altered interactomes in disease states, potentially revealing dysregulated pathways. Phosphoproteomic analysis comparing wild-type and mutant conditions can comprehensively identify substrates with altered phosphorylation states, pointing to disrupted signaling networks. In animal models, conditional knockout or knockdown of PP2A-alpha in specific neuronal populations using Cre-driver lines allows temporal and spatial investigation of its function. Behavioral phenotyping of these models, coupled with electrophysiological recordings and dendritic spine morphology analysis, can connect molecular alterations to circuit-level dysfunction and behavioral outcomes. Time-course studies during neurodevelopment are particularly important, as PP2A functions may vary across developmental stages, with early disruptions potentially leading to lasting neurological consequences .

What techniques can researchers employ to study PP2A-alpha's role in cell cycle regulation and cancer?

Investigating PP2A-alpha's role in cell cycle regulation and cancer requires multi-faceted approaches. Researchers should begin with expression analysis across diverse cancer cell lines and patient samples using validated antibodies, comparing PP2A-alpha levels in matched tumor and normal tissues through immunohistochemistry and Western blotting . Cell synchronization experiments followed by time-course analysis of PP2A-alpha activity and localization throughout the cell cycle provide insights into temporal regulation. Flow cytometry combined with phospho-specific antibodies against PP2A substrates like WEE1 can correlate phosphatase activity with cell cycle progression at the single-cell level . For functional studies, inducible knockdown or overexpression systems allow temporal control when studying a phosphatase whose complete loss may be lethal. Analysis should extend beyond proliferation to examine effects on anchorage-independent growth, migration, invasion, and sensitivity to chemotherapeutic agents. PP2A-alpha's interaction with the G2/M checkpoint can be specifically investigated by treating cells with DNA-damaging agents followed by mitotic entry assessment using phospho-histone H3 staining . Mass spectrometry-based phosphoproteomics comparing control and PP2A-alpha-depleted cells treated with cell cycle inhibitors can comprehensively identify cell cycle-specific substrates. Additionally, researchers should examine post-translational modifications of PP2A-alpha itself, including phosphorylation at Tyr307 and methylation at the C-terminal leucine, which regulate its activity and are often dysregulated in cancer .

How can researchers design experiments to investigate the interplay between PP2A-alpha and kinase signaling pathways?

Investigating the interplay between PP2A-alpha and kinase signaling pathways requires experimental designs that capture the dynamic, bidirectional nature of phosphorylation-dephosphorylation cycles. Researchers should begin with systematic pathway mapping using phospho-specific antibodies against key nodes in relevant signaling cascades (such as RAF/MEK/ERK or PI3K/AKT) in control versus PP2A-alpha-depleted cells . Temporal dynamics are critical—time-course experiments following pathway stimulation (e.g., with growth factors) in the presence or absence of PP2A-alpha can reveal which nodes are preferentially regulated by this phosphatase. Pharmacological approaches using specific kinase inhibitors in combination with PP2A modulation (either inhibition with okadaic acid/calyculin A or activation with SMAP compounds) can dissect pathway interdependencies . For direct substrate identification, researchers can employ substrate-trapping approaches using catalytically inactive PP2A-alpha mutants, which bind but fail to dephosphorylate substrates, followed by mass spectrometry to identify trapped proteins. Proximity-dependent biotinylation (BioID or TurboID) with PP2A-alpha as the bait can reveal spatially-restricted interactions with kinases and their substrates. FRET-based biosensors for specific phosphorylation events enable real-time visualization of phosphatase-kinase dynamics in living cells with subcellular resolution. Mathematical modeling incorporating measured kinetic parameters for both kinases and PP2A can predict emergent properties of these signaling networks, with experimental validation through targeted perturbations confirming model predictions . Finally, correlation analyses between PP2A-alpha expression levels and phosphorylation status of kinase pathway components across patient samples can reveal clinically relevant relationships with potential diagnostic or therapeutic implications .

What approaches can researchers use to study the structural biology of PP2A using antibodies?

Studying PP2A structural biology using antibodies requires specialized approaches that leverage these reagents beyond traditional applications. Researchers can employ antibodies as crystallization chaperones to facilitate X-ray crystallography studies—Fab fragments derived from PP2A-alpha antibodies can bind and stabilize specific conformations, potentially revealing novel structural features not captured in previous crystal structures . Cryo-electron microscopy (cryo-EM) combined with antibody labeling offers another powerful approach, where antibodies serve as fiducial markers that help identify specific regions within the three-dimensional density map, particularly valuable for localizing the catalytic subunit within the holoenzyme complex. For analyzing conformational changes, hydrogen-deuterium exchange mass spectrometry (HDX-MS) in the presence or absence of specific antibodies can reveal regions where antibody binding alters solvent accessibility, providing insights into dynamic structural elements . Antibodies can also be used in functional studies correlating structure with activity—by mapping epitopes recognized by function-modulating antibodies, researchers can identify regulatory interfaces in the PP2A complex. Site-directed mutagenesis of specific residues within identified epitopes, followed by binding and activity assays, can confirm the structural basis for functional regulation. Additionally, researchers can employ antibodies recognizing specific post-translational modifications (such as Y307 phosphorylation or C-terminal leucine methylation) to isolate and structurally characterize PP2A-alpha subpopulations with distinct modifications, potentially revealing how these modifications alter enzyme conformation and activity .

How can researchers effectively investigate PP2A-alpha in different subcellular compartments?

Investigating PP2A-alpha in different subcellular compartments requires specialized approaches for isolation, visualization, and functional analysis of spatially distinct phosphatase pools. For biochemical fractionation, researchers should employ protocols that cleanly separate cytoplasmic, nuclear, mitochondrial, and membrane compartments, followed by Western blotting with PP2A-alpha antibodies to quantify distribution. Purity controls using compartment-specific markers (lamin A/C for nucleus, GAPDH for cytoplasm, COX IV for mitochondria) are essential to confirm fractionation quality . For visualization, confocal microscopy with co-staining for organelle markers provides spatial context, while super-resolution techniques offer enhanced detail for subcompartmental localization. Live-cell imaging using fluorescently tagged PP2A-alpha (ensuring tags don't disrupt localization or function) enables dynamic tracking of phosphatase redistribution in response to stimuli . To study function in specific compartments, researchers can employ targeted PP2A-alpha constructs fused to localization sequences (nuclear localization signal, mitochondrial targeting sequence, membrane anchors) and assess local substrate dephosphorylation. Proximity labeling approaches like BioID with compartment-targeted PP2A-alpha constructs can reveal location-specific interactomes. For mechanistic understanding, researchers should investigate compartment-specific regulatory subunits that may dictate localization and substrate specificity, potentially through co-immunoprecipitation from isolated compartments followed by mass spectrometry . Finally, pharmacological or genetic perturbations that alter PP2A-alpha localization (such as nuclear export inhibitors like leptomycin B) can reveal functional consequences of disrupting its normal spatial distribution, linking compartmentalization to specific cellular processes and signaling outcomes .

How can researchers utilize PP2A-alpha antibodies in combination with phospho-proteomics approaches?

Integrating PP2A-alpha antibodies with phosphoproteomics creates powerful approaches for comprehensive phosphatase function analysis. Researchers should begin with immunoprecipitation using validated PP2A-alpha antibodies followed by mass spectrometry to identify associated proteins and potential substrates, with studies conducted under varying cellular conditions to capture context-dependent interactions . For direct substrate identification, quantitative phosphoproteomics comparing phosphopeptide abundance in control versus PP2A-alpha-depleted cells reveals sites with increased phosphorylation upon phosphatase removal. To enhance specificity, researchers can combine acute PP2A inhibition (using okadaic acid at concentrations specific for PP2A) with rapid phosphoproteomic analysis, capturing immediate phosphorylation changes before compensatory mechanisms engage . Proximity-dependent labeling approaches, where BioID or TurboID is fused to PP2A-alpha, enable identification of proteins in the immediate vicinity of the phosphatase, enriching for potential substrates and regulators. For spatial resolution, researchers can combine compartment-specific PP2A-alpha immunoprecipitation with phosphoproteomics to identify substrates in distinct subcellular locations. Temporal dynamics can be captured through time-course experiments following stimulus application, revealing the sequence and kinetics of PP2A-mediated dephosphorylation events. Validation of candidate substrates should employ multiple approaches, including in vitro dephosphorylation assays with immunoprecipitated PP2A complexes and phospho-specific antibodies against selected target sites. Finally, bioinformatic analysis of identified phosphosites can reveal enriched motifs, potentially defining sequence determinants for PP2A-alpha substrate recognition and providing predictive power for identifying additional substrates .