PPP1CA Antibody

What is PPP1CA and what are its primary biological functions?

PPP1CA (Protein Phosphatase 1 Catalytic Subunit Alpha) is one of the three catalytic subunits of protein phosphatase 1 (PP1) with a molecular weight of approximately 37 kDa. It functions as a serine/threonine-specific protein phosphatase that associates with over 200 regulatory proteins to form highly specific holoenzymes which dephosphorylate hundreds of biological targets . PPP1CA is essential for cell division and participates in the regulation of glycogen metabolism, muscle contractility, and protein synthesis. Additionally, it is involved in regulating ionic conductances and long-term synaptic plasticity . This phosphatase plays a critical role in dephosphorylating substrates such as the postsynaptic density-associated Ca²⁺/calmodulin-dependent protein kinase II. As a component of the PTW/PP1 phosphatase complex, it controls chromatin structure and cell cycle progression during the transition from mitosis into interphase .

How does PPP1CA differ from other PP1 catalytic subunits?

Four isoforms of PP1 have been characterized: PP1α (encoded by PPP1CA), PP1δ, PP1γ1, and PP1γ2 . While these isoforms share considerable sequence homology, they exhibit distinct expression patterns and functional roles. Among the three PP1 genes, PPP1CA is the most frequently amplified in various cancers . Each isoform can interact with different regulatory proteins, resulting in distinct substrate specificities and cellular localizations. An important paralog of PPP1CA is PPP1CC . These catalytic subunits have non-redundant functions in certain contexts, as evidenced by their differential association with disease states. For example, PPP1CA has been specifically implicated in androgen receptor regulation in prostate cancer, while PPP1CC (PP1γ) has been associated with enhanced cell proliferation and poor prognosis in hepatocellular carcinoma .

What is the molecular structure and cellular distribution of PPP1CA?

PPP1CA is a 330 amino acid protein with an observed molecular weight of 37 kDa . The protein contains a catalytic domain responsible for its phosphatase activity. PPP1CA is broadly expressed across various tissues and cell types . At the subcellular level, PPP1CA can be found in multiple compartments including the nucleus, cytoplasm, and centrosomes, with its precise localization often determined by its association with regulatory subunits. The protein undergoes various post-translational modifications that can affect its localization and function. In terms of structure, PPP1CA contains a metal ion-binding site essential for its catalytic activity, and a C-terminal region that participates in regulatory protein interactions .

What types of PPP1CA antibodies are available for research applications?

Research-grade PPP1CA antibodies are available in several forms including monoclonal and polyclonal variants with different host species. Monoclonal antibodies, such as the mouse anti-human monoclonal (clone P4G3AT), offer high specificity and consistency between lots . Polyclonal antibodies, like rabbit polyclonal variants, provide broad epitope recognition which can be beneficial for certain applications . The immunogens used for antibody production typically include synthetic peptides corresponding to specific regions of human PPP1CA (such as amino acids 250 to C-terminus or amino acids 30-299) . These antibodies have been validated for various applications including Western blot (WB), immunohistochemistry on paraffin-embedded sections (IHC-P), enzyme-linked immunosorbent assay (ELISA), and immunoprecipitation (IP) .

What are the optimal conditions for using PPP1CA antibodies in Western blot analyses?

For Western blot applications, PPP1CA antibodies typically perform optimally at dilutions ranging from 1:500 to 1:50,000, with a recommended starting dilution of 1:1,000 . The expected band size for PPP1CA is approximately 37 kDa. Positive controls that have been validated include HeLa cells, human brain tissue, mouse brain tissue, rat brain tissue, pig brain tissue, zebrafish tissue, T-47D cells, Jurkat cells, and HEK-293 cells . For sample preparation, standard protein extraction protocols using RIPA or NP-40 based lysis buffers are generally effective. Proper blocking (typically using 5% non-fat dry milk or BSA) and appropriate secondary antibody selection based on the primary antibody host species are essential for achieving clean, specific signals with minimal background .

How should researchers validate the specificity of PPP1CA antibodies?

Validation of PPP1CA antibody specificity is crucial due to the high sequence homology between PP1 isoforms. A comprehensive validation approach should include:

• Positive and negative control samples: Using tissues or cell lines with known PPP1CA expression levels

• Knockdown/knockout validation: Testing antibody specificity using PPP1CA-depleted samples (via siRNA, shRNA, or CRISPR-Cas9)

• Cross-reactivity assessment: Evaluating potential cross-reactivity with other PP1 isoforms

• Multiple antibody comparison: Using antibodies targeting different epitopes of PPP1CA

• Multiple detection methods: Confirming results using different techniques (WB, IHC, IP)

Researchers should also consult published literature where specific antibody clones have been validated. For instance, some PPP1CA antibodies have been extensively tested in multiple species including human, mouse, rat, pig, zebrafish, and rabbit samples .

What considerations are important when designing experiments to study PPP1CA function?

When designing experiments to investigate PPP1CA function, researchers should consider:

• Holoenzyme complexity: PPP1CA associates with over 200 regulatory proteins that determine its substrate specificity and localization

• Isoform specificity: Discriminating between PP1 isoforms requires careful selection of antibodies and genetic tools

• Cell type considerations: PPP1CA function may vary between cell types due to different regulatory protein expression

• Subcellular localization: PPP1CA functions differently depending on its localization (nuclear vs. cytoplasmic)

• Post-translational modifications: Modifications can alter PPP1CA activity and should be monitored

• Appropriate controls: Include phosphatase inhibitors (like okadaic acid or calyculin A) at concentrations specific for PPP1CA inhibition

• Temporal dynamics: PPP1CA activity changes throughout the cell cycle and in response to various stimuli

Experimental approaches should combine biochemical assays (phosphatase activity measurements), genetic manipulations (knockdown/knockout), and cell biological techniques (localization studies) for comprehensive analysis.

How can researchers effectively measure PPP1CA phosphatase activity?

Measuring PPP1CA phosphatase activity requires careful consideration of several factors:

• Substrate selection: Use physiologically relevant phosphorylated proteins or peptides that are known PPP1CA substrates

• Isolation strategies: Immunoprecipitate PPP1CA using specific antibodies before activity assays to separate it from other phosphatases

• Detection methods:

  • Colorimetric assays measuring phosphate release (e.g., malachite green assay)

  • Radioactive assays using ³²P-labeled substrates

  • Fluorescent methods using phospho-sensitive fluorescent probes

  • Western blotting with phospho-specific antibodies to detect substrate dephosphorylation

• Regulatory protein consideration: Reconstitute PPP1CA with specific regulatory subunits to assess holoenzyme activity

• Controls: Include phosphatase inhibitors at appropriate concentrations to confirm specificity

• Normalization: Normalize activity to the amount of PPP1CA protein to distinguish between changes in expression versus specific activity

This multi-faceted approach allows for robust measurement of PPP1CA activity under various experimental conditions.

What approaches can be used to study PPP1CA-protein interactions?

To investigate PPP1CA interactions with regulatory proteins and substrates, researchers can employ:

• Co-immunoprecipitation: Using PPP1CA antibodies to pull down protein complexes, followed by Western blotting or mass spectrometry to identify interactors

• Yeast two-hybrid screening: To identify direct protein-protein interactions

• Proximity labeling approaches: BioID or APEX to identify proteins in close proximity to PPP1CA in living cells

• Fluorescence techniques: FRET or BiFC to visualize protein interactions in live cells

• Structural approaches: X-ray crystallography or cryo-electron microscopy to determine 3D structures of PPP1CA-protein complexes

• Peptide arrays: To map interaction interfaces and binding motifs

• Pull-down assays: Using recombinant PPP1CA as bait to identify interacting proteins

• Competitive binding assays: To determine if regulatory proteins compete for binding to PPP1CA

These complementary approaches provide a comprehensive view of the PPP1CA interactome in different cellular contexts.

How is PPP1CA implicated in cancer biology?

PPP1CA plays significant roles in cancer development and progression through multiple mechanisms:

• Gene amplification: PPP1CA is frequently amplified in various cancers, particularly prostate cancer

• Androgen receptor regulation: PPP1CA suppresses androgen receptor ubiquitylation and degradation, thereby enhancing receptor stability and activity in prostate cancer

• Therapy resistance: PPP1CA contributes to docetaxel resistance in prostate cancer by upregulating Caprin1-dependent stress granule assembly

• Cell cycle control: As a regulator of cell division, PPP1CA dysregulation can contribute to uncontrolled proliferation

• Apoptosis regulation: PPP1CA modulates phosphorylation status of apoptotic proteins

• Metastasis: Expression of EMT-related genes like CAMK2N1 and WNT5A is increased in locally invasive and metastatic prostate cancer, with PPP1CA involved in their regulation

Experimental approaches to study PPP1CA in cancer include analysis of gene expression databases, immunohistochemical staining of tumor samples, functional studies in cancer cell lines, and analysis of patient-derived xenografts .

What role does PPP1CA play in neurological function and disorders?

PPP1CA contributes to neurological function and has been implicated in several neurological processes:

• Synaptic plasticity: PPP1CA regulates long-term synaptic plasticity and is involved in dephosphorylating postsynaptic density-associated Ca²⁺/calmodulin-dependent protein kinase II

• Learning and memory: Mouse studies suggest that PP1 functions as a suppressor of learning and memory processes

• Neural development: PPP1CA regulates neural tube and optic fissure closure, and enteric neural crest cell migration during development

• Circadian rhythm regulation: In balance with CSNK1D and CSNK1E, PPP1CA determines circadian period length through regulation of PER1 and PER2 phosphorylation

• Intellectual disability: De novo mutations in PPP1CB (a related PP1 catalytic subunit) are associated with moderate to severe intellectual disability, suggesting potential roles for PP1 family members in neurodevelopmental disorders

Experimental approaches include electrophysiological recordings, behavioral testing in animal models, phosphoproteomic analysis of neural tissues, and genetic association studies in patients with neurological disorders .

How is PPP1CA involved in cardiac function and heart failure?

PPP1CA has significant implications for cardiac function and heart failure:

• Increased activity in heart failure: Increased PP1 activity has been detected in the end stage of heart failure

• Cardiac function regulation: Studies in both humans and mice suggest that PP1 is a significant regulator of cardiac function

• Contractility modulation: PPP1CA regulates the phosphorylation status of proteins involved in cardiac muscle contractility

• Calcium handling: PPP1CA influences calcium cycling in cardiomyocytes through dephosphorylation of key calcium handling proteins

• Congenital heart disease: De novo variants in PPP1CB (a related PP1 catalytic subunit) are associated with congenital heart disease, suggesting potential roles for PP1 family members in cardiac development

Research approaches include analysis of PPP1CA expression and activity in heart failure models, genetic manipulation in animal models, functional studies in isolated cardiomyocytes, and correlation studies in patient samples .

What are common challenges when working with PPP1CA antibodies and how can they be overcome?

Researchers commonly encounter these challenges when working with PPP1CA antibodies:

• Cross-reactivity with other PP1 isoforms: Due to high sequence homology between PP1 catalytic subunits

  • Solution: Use antibodies raised against unique regions and validate with isoform-specific controls or knockout samples

• Non-specific binding and high background:

  • Solution: Optimize blocking conditions (5% BSA or milk), antibody dilutions (1:5000-1:50000 for WB), and washing steps; consider using more specific monoclonal antibodies

• Variability in antibody performance across applications:

  • Solution: Different antibody clones may perform better in specific applications; test multiple antibodies and optimize conditions for each application

• Detection of regulatory subunit-bound vs. free PPP1CA:

  • Solution: Use native conditions for some experiments to preserve complexes and denaturing conditions for others to detect total PPP1CA

• Storage and stability issues:

  • Solution: Store antibodies at -20°C for long-term storage and at 4°C for up to one month; prevent freeze-thaw cycles by preparing small aliquots

• Species cross-reactivity limitations:

  • Solution: Verify cross-reactivity claims with proper controls; consider species-specific antibodies for certain applications

How can researchers differentiate between the activities of different PP1 isoforms in experimental systems?

Differentiating between PP1 isoform activities requires strategic experimental approaches:

• Isoform-specific antibodies: Use rigorously validated antibodies that specifically recognize each isoform without cross-reactivity

• Genetic manipulation:

  • Use siRNA or shRNA targeting unique regions (often 3' UTRs) of each isoform

  • CRISPR-Cas9 knockout of specific isoforms followed by rescue experiments

  • Isoform-specific promoter-reporter constructs to study differential regulation

• Recombinant protein studies:

  • Express and purify individual isoforms for in vitro activity assays

  • Create chimeric proteins to identify functional domains specific to each isoform

• Tissue and cell-type analysis:

  • Leverage natural differences in isoform expression across tissues

  • Single-cell approaches to identify cell types with differential isoform expression

• Isoform-specific interactors:

  • Identify proteins that interact specifically with one isoform

  • Use these interactions as proxies for isoform-specific functions

• Specific inhibitors:

  • When available, use inhibitors with selectivity between isoforms

This multi-faceted approach allows researchers to assign specific functions to each PP1 catalytic subunit.

What statistical approaches are recommended for analyzing PPP1CA expression and activity data?

Robust statistical analysis of PPP1CA data should include: