PPP2R3A Antibody, Biotin conjugated

What is PPP2R3A and where is it primarily expressed?

PPP2R3A (Protein Phosphatase 2, Regulatory Subunit B'', alpha) is predominantly expressed in the cytoplasm of cells, with some expression also detected on cell membranes. In hepatocellular carcinoma (HCC) tissues, PPP2R3A protein shows significantly higher expression compared to adjacent normal tissues, indicating its potential role in cancer development. Immunohistochemical studies have confirmed this cytoplasmic localization pattern, which appears critical to the biological function of this protein. The PR130 subtype of PPP2R3A is known to redistribute SH2-containing inositol phosphatase 2 in the cell membrane and inhibit the degradation of epidermal growth factor receptor after binding to it . Understanding this cellular distribution is essential for designing appropriate experimental protocols when using PPP2R3A antibodies.

What are the available binding specificities for PPP2R3A antibodies?

Several PPP2R3A antibodies are available with different binding specificities targeting various amino acid regions of the protein. Common binding specificities include antibodies recognizing AA 851-940, AA 801-900, AA 179-228, and AA 256-508 regions of the PPP2R3A protein . These different binding specificities allow researchers to target specific domains of the protein depending on experimental needs. For instance, when studying protein-protein interactions or specific functional domains, selecting an antibody that targets the relevant amino acid region is crucial for obtaining meaningful results. Researchers should carefully consider which epitope region would be most appropriate for their specific research question before selecting an antibody.

What are the main applications for biotin-conjugated PPP2R3A antibodies?

Biotin-conjugated PPP2R3A antibodies are versatile tools suitable for multiple applications, including Western Blotting (WB), ELISA, Immunohistochemistry on both paraffin-embedded (IHC-p) and frozen (IHC-fro) sections . The biotin conjugation provides amplification of signal and flexibility in detection systems, making these antibodies particularly valuable for detecting low-abundance proteins or when high sensitivity is required. The biotin-streptavidin system offers one of the strongest non-covalent biological interactions known, providing stable and specific detection capabilities. This conjugation also facilitates compatibility with various detection methods, allowing researchers to optimize visualization strategies according to their specific experimental setup and available equipment.

What species reactivity can be expected with commercially available PPP2R3A antibodies?

Commercial PPP2R3A antibodies, including biotin-conjugated versions, typically show confirmed reactivity with human samples, with many also cross-reacting with mouse and rat tissues . Additional predicted reactivity often includes dog, horse, chicken, and rabbit, although these should be experimentally validated before use in critical experiments. When working with less common research organisms, researchers should either validate the antibody's cross-reactivity themselves or contact manufacturers for additional information regarding potential cross-reactivity. For studies involving multiple species comparisons, selecting antibodies with confirmed multi-species reactivity is advisable to ensure consistent experimental conditions across different samples.

How should researchers optimize immunohistochemistry protocols for PPP2R3A detection?

For optimal immunohistochemical detection of PPP2R3A, researchers should follow a structured approach to protocol development. Based on published methodologies, tissue specimens should be formalin-fixed, paraffin-embedded, and sectioned to approximately 4-μm thickness . A two-step immunohistochemical detection method is recommended, using PPP2R3A primary antibody at a dilution of 1:200 to 1:500 . For biotin-conjugated antibodies, ensure appropriate blocking of endogenous biotin to prevent background staining, particularly in biotin-rich tissues such as liver. Antigen retrieval methods should be optimized depending on fixation conditions, with citrate buffer (pH 6.0) heat-induced epitope retrieval often yielding good results. Detection can be accomplished using streptavidin-conjugated enzyme systems, with careful optimization of incubation times to maximize signal-to-noise ratio.

What controls should be implemented when using PPP2R3A antibodies in cancer research?

Implementing proper controls is crucial for generating reliable data with PPP2R3A antibodies in cancer research. Negative controls should include omission of primary antibody, substituting it with non-immune serum from the same species as the primary antibody (e.g., rabbit serum for rabbit-derived PPP2R3A antibodies) . Positive controls should include tissues known to express PPP2R3A, such as HCC samples previously validated for PPP2R3A expression. Additionally, comparing expression between tumor tissues and adjacent normal tissues provides an internal control and reference for expression levels. For biotin-conjugated antibodies specifically, additional controls should include streptavidin-only conditions to rule out non-specific binding. When evaluating the functional significance of PPP2R3A, both overexpression and knockdown/knockout approaches should be employed to demonstrate specificity of observed effects.

How can researchers quantify PPP2R3A expression in tissue samples?

Quantification of PPP2R3A expression in tissue samples can be performed using a semi-quantitative scoring system that combines staining intensity and proportion of positive cells. One validated approach uses a 0-3 scale for staining intensity (0 = no staining, 1 = light yellow, 2 = brown, 3 = tan) and a 0-3 scale for percentage of positive cells (0 = <5%, 1 = 5-25%, 2 = 26-50%, 3 = >50%) . The final score is calculated by multiplying these values, resulting in scores ranging from 0-9, with scores ≥4 typically classified as high expression and <4 as low expression. This scoring system has demonstrated clinical relevance in distinguishing prognostic groups in HCC patients after liver transplantation. For more objective quantification, digital image analysis using specialized software can provide continuous measurements of staining intensity and area, reducing observer bias.

How is PPP2R3A expression correlated with clinical parameters in hepatocellular carcinoma?

High expression of PPP2R3A in HCC tissues has shown significant correlation with several important clinical parameters. Research demonstrates that elevated PPP2R3A expression positively correlates with preoperative serum alpha-fetoprotein (AFP) levels (P = 0.003), tumor-node-metastasis-t stage (P ≤ 0.001), and envelope invasion (P = 0.001) . These correlations suggest PPP2R3A involvement in more aggressive disease phenotypes. When designing experiments to investigate PPP2R3A in HCC models, researchers should consider stratifying samples based on these clinical parameters to identify potential mechanisms underlying these associations. Additionally, collecting comprehensive clinical data alongside PPP2R3A expression analysis is recommended to enable robust correlation studies and identification of potential confounding factors that might influence experimental outcomes.

What is the functional significance of PPP2R3A in cancer cell biology?

Studies have revealed that PPP2R3A overexpression promotes proliferation of HCC cells and potentially enhances invasion and migration capabilities . Conversely, down-regulation of PPP2R3A gene expression inhibits HCC cell proliferation, suggesting its oncogenic role in liver cancer. Mechanistically, the PR130 subtype of PPP2R3A has been shown to redistribute SH2-containing inositol phosphatase 2 in the cell membrane and inhibit degradation of epidermal growth factor receptor (EGFR), which has established tumor-promoting effects across multiple cancer types. When studying cancer cell behavior in relation to PPP2R3A, researchers should design experiments that evaluate these specific phenotypes (proliferation, invasion, migration) and consider investigating the interaction between PPP2R3A and EGFR signaling pathways to elucidate the molecular mechanisms involved.

What are common issues when using biotin-conjugated PPP2R3A antibodies and how can they be resolved?

Several technical challenges can arise when working with biotin-conjugated PPP2R3A antibodies. One common issue is high background staining due to endogenous biotin, particularly in biotin-rich tissues such as liver, kidney, and brain. This can be addressed by implementing an avidin-biotin blocking step prior to primary antibody application. Non-specific binding can also occur, resulting in false-positive signals. To minimize this, researchers should optimize blocking conditions using appropriate blocker (typically 5-10% normal serum from the same species as the secondary antibody). Insufficient signal is another frequent problem, which may be remedied by adjusting antibody concentration, extending incubation time, or enhancing antigen retrieval methods. For Western blotting applications specifically, optimization of transfer conditions for high-molecular-weight proteins like PPP2R3A (approximately 130 kDa) is crucial for efficient transfer and detection.

How can researchers validate the specificity of PPP2R3A antibodies?

Validating antibody specificity is crucial for generating reliable research data. For PPP2R3A antibodies, researchers should implement a multi-faceted validation approach. First, compare staining patterns across multiple antibodies targeting different epitopes of PPP2R3A to confirm consistent localization patterns . Second, perform knockdown or knockout experiments using siRNA or CRISPR-Cas9 systems to verify that the signal diminishes with reduced PPP2R3A expression. Third, conduct peptide competition assays using the immunizing peptide to confirm specific binding. Western blotting should show a single band at the expected molecular weight (~130 kDa for full-length PPP2R3A), though alternative splicing variants may produce additional bands. For biotin-conjugated antibodies specifically, comparing the staining pattern with unconjugated versions of the same antibody clone can help confirm that the conjugation process hasn't altered specificity.

How can PPP2R3A antibodies be utilized in combination with other protein phosphatase antibodies for pathway analysis?

For comprehensive phosphatase pathway analysis, PPP2R3A antibodies can be strategically combined with antibodies targeting other protein phosphatase components. Since PPP2R3A is a regulatory subunit of protein phosphatase 2A (PP2A), researchers should consider multiplex staining approaches that simultaneously detect catalytic subunits (PP2A-C), structural subunits (PP2A-A), and other regulatory subunits. This provides insights into the complete holoenzyme composition across different cellular contexts. Co-immunoprecipitation experiments using PPP2R3A antibodies can identify novel interaction partners within phosphatase complexes. For signaling pathway analysis, combining PPP2R3A detection with phospho-specific antibodies for downstream targets can elucidate the functional consequences of PPP2R3A expression alterations. When designing such experiments, careful selection of compatible antibodies (considering species origin, isotype, and conjugation) is essential for successful multiplexed detection approaches.

What are emerging applications for PPP2R3A antibodies in cancer biomarker research?

Emerging research suggests promising applications for PPP2R3A antibodies in cancer biomarker development. Given the demonstrated correlation between high PPP2R3A expression and poor prognosis in HCC patients after liver transplantation , antibodies against this protein could be valuable components of immunohistochemical biomarker panels for patient stratification. Researchers are exploring the combination of PPP2R3A expression with other markers, such as AFP levels, to create more precise prognostic tools. Potential applications include development of tissue microarray-based screening approaches for rapid assessment of PPP2R3A status across large patient cohorts. Additionally, liquid biopsy applications may emerge, focusing on detecting PPP2R3A in circulating tumor cells or exosomes. When designing biomarker validation studies, researchers should adhere to REMARK (REporting recommendations for tumor MARKer prognostic studies) guidelines to ensure clinical utility of their findings.