ppp2r3c Antibody

What is PPP2R3C and why is it important for research?

PPP2R3C (also known as C14orf10, G5PR, Protein phosphatase subunit G5PR, and Rhabdomyosarcoma antigen MU-RMS-40.6A/6C) encodes a serine/threonine-protein phosphatase 2A regulatory subunit that plays multiple critical roles in cellular function. It regulates MCM3AP phosphorylation through phosphatase recruitment, acts as a negative regulator of ABCB1 expression via the TFPI2/PPP2R3C complex, and contributes to activation-induced cell death in B-cells . Recent research has also identified PPP2R3C as having centrosomal localization with roles in microtubule organization and cell signaling . Additionally, novel compound heterozygous variants in PPP2R3C have been linked to syndromic 46, XY gonadal dysgenesis, highlighting its importance in developmental biology . These diverse functions make PPP2R3C antibodies valuable tools for studying phosphatase regulation, immune cell development, and cellular architecture.

What types of PPP2R3C antibodies are available for research applications?

Currently, researchers have access to several types of PPP2R3C antibodies optimized for different experimental applications. The most common are rabbit polyclonal antibodies that target different epitopes of human PPP2R3C. These include antibodies targeting the 165-214 amino acid region and those raised against recombinant protein of human PPP2R3C . Most commercially available antibodies are unconjugated with IgG isotypes and demonstrate cross-reactivity with human, mouse, and rat samples . Both affinity-purified antibodies and non-purified formats are available, with the purified versions offering higher specificity for sensitive applications like immunohistochemistry. For specialized applications, researchers may also use tagged PPP2R3C constructs (such as PPP2R3C-GFP) for transgenic expression and visualization in live cells .

What are the recommended applications for PPP2R3C antibodies?

PPP2R3C antibodies have been validated for multiple research applications with varying recommended protocols:

When designing experiments, researchers should note that PPP2R3C shows different subcellular localizations depending on cell cycle stage, being present in both nucleus and cytoplasm but excluded from nucleoli, with cytoplasmic localization during cytokinesis .

How should I optimize PPP2R3C antibody validation for my specific cell line or tissue?

When validating PPP2R3C antibodies for new experimental systems, a multi-step approach is recommended. First, assess endogenous expression levels of PPP2R3C in your model system through reference databases or preliminary RT-PCR analysis. Cell lines with high endogenous expression, such as SKNBE2 neuroblastoma cells, may provide stronger signals for initial validation . For antibody validation, implement a hierarchical testing strategy:

• Begin with Western blot analysis using positive control samples alongside your experimental system, looking for a specific band at the expected molecular weight.

• Include negative controls, ideally using PPP2R3C knockout cells if available. CRISPR-based PPP2R3C knockout models have been developed and can serve as excellent specificity controls .

• For immunocytochemistry applications, compare antibody staining patterns with the known subcellular distribution (nuclear, cytoplasmic, excluded from nucleoli) and verify cell-cycle dependent localization patterns .

• If possible, use orthogonal detection methods such as a PPP2R3C-GFP construct to confirm localization patterns observed with the antibody .

• For challenging applications like ultra-structure expansion microscopy, where traditional antibody staining may be compromised after gelation and denaturation, consider using transgene approaches with PPP2R3C-GFP and anti-GFP antibodies .

Additionally, testing antibody specificity across multiple dilutions can help identify optimal signal-to-noise ratios for your specific experimental system.

What are the critical storage and handling requirements for maintaining PPP2R3C antibody efficacy?

Long-term stability and performance of PPP2R3C antibodies depend significantly on proper storage and handling practices. Most commercial PPP2R3C antibodies are supplied in a liquid format buffered in PBS with stabilizers such as 50% glycerol and preservatives like 0.05% sodium azide . For optimal antibody performance:

• Store antibodies at -20°C for long-term storage, with an expected shelf-life of approximately one year from receipt when properly maintained .

• Critically, avoid repeated freeze-thaw cycles which can lead to antibody degradation and loss of binding efficacy. If frequent usage is anticipated, consider aliquoting the antibody into single-use volumes upon receipt .

• When removing antibodies from storage, thaw completely at 4°C before use and maintain cold chain throughout handling.

• Prior to application, centrifuge briefly to collect solution at the bottom of the tube and mix gently to ensure homogeneity.

• For immunohistochemistry applications requiring higher sensitivity, prepare working dilutions immediately before use rather than storing diluted antibody solutions.

• Monitor for signs of contamination or precipitation, which may indicate compromised antibody quality.

Researchers should note that buffer compositions containing BSA (0.5%) and sodium azide (0.02-0.05%) are not compatible with certain applications such as live cell imaging or functional assays where cell viability is essential .

How can I troubleshoot weak or non-specific signals when using PPP2R3C antibodies?

When encountering weak or non-specific signals with PPP2R3C antibodies, systematic troubleshooting is essential. Common issues and remediation strategies include:

For weak signals:

• Verify PPP2R3C expression levels in your experimental system, as expression can vary significantly between cell types. Consider using positive controls such as SKNBE2 neuroblastoma cells that have high endogenous PPP2R3C expression .

• Adjust antibody concentration by testing a range of dilutions beyond the recommended range (e.g., 1:50-1:500 for IHC applications) .

• Optimize antigen retrieval methods for fixed tissues, as PPP2R3C epitopes may be partially masked during fixation.

• Extend primary antibody incubation time or switch to overnight incubation at 4°C to enhance binding.

• For Western blot applications, ensure adequate protein loading (50-100 μg total protein) and consider using enhanced chemiluminescence detection systems.

For non-specific signals:

• Increase blocking stringency using 5% BSA or 10% normal serum from the same species as the secondary antibody.

• Validate antibody specificity using PPP2R3C knockout cell lines as negative controls .

• For immunofluorescence applications, high background may be reduced by additional washing steps and inclusion of 0.1-0.3% Triton X-100 in washing buffers.

• Consider secondary antibody-only controls to rule out non-specific binding of the detection system.

• For specialized applications like ultra-structure expansion microscopy, standard antibody staining may yield punctate patterns that appear non-specific; in such cases, transgene approaches (e.g., PPP2R3C-GFP) may provide more robust labeling .

How can PPP2R3C antibodies be utilized to study its role in centrosome function and cell division?

Recent research has revealed PPP2R3C localization at centrosomes, suggesting important roles in microtubule organization and cell signaling. To effectively study these functions:

• Ultra-structure expansion microscopy (U-ExM) with anti-PPP2R3C antibodies or PPP2R3C-GFP constructs can resolve the protein's precise localization at centrioles. This technique has revealed that PPP2R3C forms a cylindrical distribution at the distal end of centrioles with a diameter of approximately 300 nm .

• Co-immunoprecipitation coupled with mass spectrometry using PPP2R3C antibodies can identify centrosome-associated protein interaction partners. Pull-down assays followed by Western blotting can validate specific interactions with known centrosomal components.

• For dynamic studies, researchers can implement live-cell imaging with PPP2R3C-GFP to track protein redistribution throughout the cell cycle, particularly during transitions between nuclear localization and cytoplasmic accumulation during cytokinesis .

• Cell proliferation assays comparing wild-type cells to PPP2R3C knockouts can quantify the impact of PPP2R3C loss on cell division rates. Previous studies have documented significant growth defects in PPP2R3C-knockout SKNBE2 neuroblastoma cells that could be rescued by expression of an sgRNA-resistant PPP2R3C-GFP transgene .

• Combined immunofluorescence with PPP2R3C antibodies and established centrosomal markers can reveal colocalization patterns and potential functional relationships during different cell cycle stages.

What approaches are recommended for studying PPP2R3C mutations and their impact on protein function?

The discovery of disease-associated PPP2R3C variants, particularly those linked to syndromic 46, XY gonadal dysgenesis, has opened new avenues for functional research. To effectively investigate PPP2R3C mutations:

• Generate cell models expressing specific PPP2R3C variants such as c.684_686delTTC/p.F229del and c.1250G>A/p.G417E through CRISPR-Cas9 knock-in techniques or transfection of mutant constructs into PPP2R3C-null backgrounds .

• Analyze structural changes using computational modeling to predict how mutations affect protein folding and function. For instance, the deletion of Phe229 has been shown to cause an incomplete alpha helix structure and disrupt four repeated phenylalanines, while the Gly417Glu mutation alters hydrogen bonding patterns within the protein .

• Assess phosphatase regulatory activity through in vitro phosphatase assays comparing wild-type and mutant PPP2R3C proteins, particularly focusing on known substrates such as MCM3AP and ABCB1 .

• Evaluate protein-protein interactions using co-immunoprecipitation with PPP2R3C antibodies to determine if mutations disrupt binding to regulatory partners. Cross-linking approaches can stabilize transient interactions for more comprehensive interaction mapping.

• For mutations affecting B-cell function, flow cytometry analysis of lymphocyte populations can quantify changes in CD19+ B cells and CD4+ T cells. Gene knockout studies in mice have demonstrated that PPP2R3C is essential for B-cell survival, and human patients with PPP2R3C mutations show decreased CD19+ B cell populations (1.6% compared to normal range of 8.5-14.5%) .

How can I effectively use PPP2R3C antibodies to investigate its role in B-cell development and immune function?

PPP2R3C plays a crucial role in B-cell survival and immune regulation, making antibodies against this protein valuable tools for immunological research:

• For flow cytometry applications, combine PPP2R3C antibody staining with B-cell surface markers (CD19, CD20) to correlate PPP2R3C expression levels with B-cell maturation stages. Research has shown that PPP2R3C deficiency results in significantly reduced mature B-cell populations .

• In tissue sections from lymphoid organs, immunohistochemistry with PPP2R3C antibodies can reveal spatial distribution patterns within germinal centers and other B-cell rich regions. Use dilutions of 1:50-300 for optimal staining intensity in fixed tissues .

• To study activation-induced cell death mechanisms, stimulate B-cells with activating agents (anti-IgM, CD40L) and monitor changes in PPP2R3C subcellular localization using immunofluorescence. The protein has been implicated in the regulation of B-cell apoptosis following activation signals .

• For mechanistic studies, combine PPP2R3C immunoprecipitation with phosphoproteomic analysis to identify substrates specifically dephosphorylated in B-cells following activation.

• When investigating human samples, consider the quantitative analysis of lymphocyte subsets in individuals with PPP2R3C variants. Previous research documented decreased CD19+ B-cells (1.6%, compared to normal range of 8.5%-14.5%) and reduced CD4+ T-cells (21.5%, normal range: 30.0-46.0%) in patients with PPP2R3C mutations .

• For functional validation, use CRISPR-mediated PPP2R3C knockout in B-cell lines followed by rescue experiments with wild-type or mutant PPP2R3C constructs to establish direct causality between PPP2R3C activity and observed phenotypes.

What emerging techniques could enhance PPP2R3C protein research beyond current antibody capabilities?

While antibodies remain essential tools for PPP2R3C research, several emerging technologies offer complementary approaches with potential advantages:

• Proximity labeling methods such as BioID or APEX2 fused to PPP2R3C can map the protein's local interactome within specific subcellular compartments like centrosomes, potentially revealing transient or weak interactions missed by traditional co-immunoprecipitation approaches .

• CRISPR activation/inhibition (CRISPRa/CRISPRi) systems targeting PPP2R3C can provide more precise temporal control over gene expression than traditional knockout approaches, enabling the study of dose-dependent effects and acute versus chronic loss.

• Single-cell RNA-sequencing combined with protein detection (CITE-seq) could correlate PPP2R3C protein levels with transcriptional states in heterogeneous cell populations, particularly valuable for understanding its role in B-cell development and activation .

• Super-resolution microscopy techniques beyond expansion microscopy, such as STORM or PALM, may resolve PPP2R3C's nanoscale organization at centrosomes and other subcellular structures with even greater precision than currently achieved .

• Phosphoproteomics approaches comparing wild-type and PPP2R3C-deficient cells could comprehensively identify substrates regulated by PPP2R3C-containing phosphatase complexes, providing insights into its broader signaling functions.

• Patient-derived induced pluripotent stem cells (iPSCs) carrying PPP2R3C mutations offer opportunities to study developmental consequences in relevant cell lineages, particularly for understanding its role in gonadal development disorders .

How can researchers integrate PPP2R3C antibody data with other omics approaches for systems-level understanding?

To achieve a comprehensive understanding of PPP2R3C function, researchers should consider integrating antibody-based findings with multiple omics approaches:

• Combine immunoprecipitation using PPP2R3C antibodies with mass spectrometry (IP-MS) to define the protein interactome, then integrate with transcriptomic data to identify coordinated gene-protein regulatory networks.

• Correlate PPP2R3C localization data from immunofluorescence studies with chromatin accessibility maps (ATAC-seq) and histone modification patterns to understand its potential nuclear functions beyond direct phosphatase activity.

• For disease-associated PPP2R3C variants, integrate structural predictions of protein changes with phenotypic data from patient samples and model systems. This approach has already revealed connections between specific mutations (e.g., c.684_686delTTC/p.F229del and c.1250G>A/p.G417E) and syndromic features including gonadal dysgenesis and immune abnormalities .

• Implement parallel reaction monitoring (PRM) mass spectrometry to quantify PPP2R3C phosphorylation states across different cellular conditions, complementing antibody-based detection methods.

• Develop computational models incorporating protein-protein interaction data, phosphorylation dynamics, and cellular phenotypes to predict systemic consequences of PPP2R3C perturbation across different tissues and developmental stages.

• For B-cell development studies, integrate flow cytometry data showing reduced CD19+ populations in PPP2R3C-deficient conditions with single-cell transcriptomics to identify specific developmental checkpoints requiring PPP2R3C function .