PDE2A Antibody

What is PDE2A and why is it important in neuroscience research?

PDE2A (phosphodiesterase 2A, cGMP-stimulated) belongs to the cyclic nucleotide phosphodiesterase family and PDE2 subfamily. It functions as a dual-specificity enzyme that regulates both cAMP and cGMP second messenger signaling pathways, which are crucial for various cellular processes . PDE2A is particularly abundant in brain tissue compared to peripheral tissues, suggesting its significant role in neuronal function and signaling . Its importance in neuroscience stems from its involvement in regulating spatio-temporal signaling by intracellular second messengers that modulate synaptic plasticity, neuronal excitability, and network activity . Research has shown that PDE2A inhibition can enhance axonal sprouting and promote the association of neuronal activity in brain networks, particularly in cortical areas, demonstrating its potential therapeutic relevance in conditions like stroke .

What is the molecular structure and cellular localization of PDE2A?

PDE2A is a protein with a calculated molecular weight of 106 kDa, though it is typically observed at 100-106 kDa in experimental conditions . It has several conserved domains, including a catalytic domain that is critical for its phosphodiesterase activity. In terms of cellular localization within the brain, PDE2A protein is predominantly concentrated in axons and nerve terminals of neurons, with noticeably weaker expression in cell bodies and proximal dendrites . This distribution pattern suggests that PDE2A primarily functions in presynaptic regulation of neurotransmitter release rather than in postsynaptic signaling. Regional distribution studies have revealed that PDE2A mRNA is prominently expressed in glutamatergic pyramidal cells in the cortex, and in pyramidal and dentate granule cells in the hippocampus . Additionally, small populations of non-pyramidal cells, presumed to be interneurons, in both hippocampus and cortex show strong PDE2A immunoreactivity .

How do PDE2A antibodies differ across species reactivity and applications?

PDE2A antibodies are designed to recognize specific epitopes on the PDE2A protein and can vary in their reactivity across species. For instance, the PDE2A antibody (55306-1-AP) from Proteintech demonstrates validated reactivity with human, mouse, and rat samples . This cross-species reactivity is particularly valuable for comparative studies examining the conservation of PDE2A function across different experimental animal models. The pattern of PDE2A immunoreactivity has been shown to be consistent across rats, mice, cynomolgus monkeys, dogs, and humans, suggesting evolutionary conservation of this protein's expression and function .

In terms of applications, PDE2A antibodies are validated for various experimental techniques. The 55306-1-AP antibody, for example, can be used in Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Immunoprecipitation (IP), and ELISA applications . Each application requires specific optimization of antibody dilution, with recommended dilutions typically ranging from 1:500-1:2000 for Western blot, 1:20-1:200 for immunohistochemistry, and 0.5-4.0 μg for immunoprecipitation with 1.0-3.0 mg of total protein lysate .

How should I optimize PDE2A antibody dilutions for different experimental applications?

For immunohistochemistry applications, a wider dilution range of 1:20-1:200 is recommended . When performing IHC on tissue sections such as human kidney or liver cancer tissue, antigen retrieval becomes an important consideration. The suggested protocol involves using TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative option . For immunoprecipitation, 0.5-4.0 μg of antibody should be used for 1.0-3.0 mg of total protein lysate .

It is essential to remember that these recommendations serve as starting points, and the antibody should be titrated in each specific testing system to achieve optimal results. The optimal dilution may vary depending on the sample type, fixation method, detection system, and other experimental variables .

What are the best methods for validating PDE2A antibody specificity?

Validating antibody specificity is crucial for ensuring the reliability of experimental results. Several complementary approaches are recommended for comprehensive validation of PDE2A antibodies:

What sample preparation techniques optimize PDE2A detection in immunohistochemistry?

Optimal sample preparation for PDE2A detection in immunohistochemistry requires attention to several critical factors:

• Fixation: Proper tissue fixation is essential for preserving PDE2A antigenicity while maintaining tissue morphology. Paraformaldehyde fixation (typically 4%) has been successfully employed in studies examining PDE2A distribution .

• Antigen retrieval: For formalin-fixed, paraffin-embedded tissues, antigen retrieval is crucial. The recommended protocol for PDE2A antibody (55306-1-AP) suggests using TE buffer at pH 9.0, with citrate buffer at pH 6.0 as an alternative . The specific pH of the retrieval buffer is important as it can affect epitope accessibility.

• Blocking: Effective blocking of non-specific binding sites using appropriate blocking solutions (typically containing serum from the species in which the secondary antibody was raised) reduces background staining and improves signal-to-noise ratio.

• Detection systems: For visualization of PDE2A immunoreactivity, various detection methods have been employed. For instance, fluorescent secondary antibodies such as anti-rabbit Alexa 680 have been used for Western blot visualization . For colorimetric detection in immunohistochemistry, streptavidin-alkaline phosphatase and chromogenic substrates like 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium chloride (as used in in situ hybridization visualization) can be utilized .

• Controls: Including both positive controls (tissues known to express PDE2A, such as brain tissue) and negative controls (omission of primary antibody or use of tissues from PDE2A knockout models) is essential for validating staining specificity .

What is the distribution pattern of PDE2A in the mammalian brain?

PDE2A exhibits a heterogeneous distribution pattern in the mammalian brain, with significant regional and cellular specificity. At the mRNA level, PDE2A is prominently expressed in glutamatergic pyramidal cells in the cortex, and in both pyramidal and dentate granule cells in the hippocampus . This pattern is consistent across different mammalian species, including rats, mice, cynomolgus monkeys, dogs, and humans .

At the protein level, PDE2A is predominantly concentrated in the axons and nerve terminals of neurons, with markedly weaker expression in cell bodies and proximal dendrites . This subcellular distribution suggests a specialized role in regulating presynaptic functions. In the hippocampus and cortex, small populations of non-pyramidal cells, presumed to be interneurons, show strong PDE2A immunoreactivity .

This specific distribution pattern suggests that PDE2A may modulate cortical, hippocampal, and striatal networks at several levels, with a potential focus on regulating neurotransmitter release and other presynaptic functions .

How does PDE2A expression vary across different cancer types?

Recent comprehensive pan-cancer analyses have revealed significant variations in PDE2A expression across different cancer types, suggesting its potential role as a biomarker for various cancers . Analysis of data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases has shown that PDE2A levels are notably lower in several cancer types compared to their corresponding normal tissues . These cancer types include:

• Adrenocortical carcinoma (ACC)

• Acute myeloid leukemia (LAML)

• Lower-grade glioma (LGG)

• Ovarian serous cystadenocarcinoma (OV)

• Testicular germ cell tumors (TGCT)

• Uterine carcinosarcoma (UCS)

In contrast, in thymoma (THYM) tissues, PDE2A levels are significantly elevated compared to normal tissues . These differential expression patterns suggest that PDE2A may play diverse roles in different cancer types, potentially functioning as either a tumor suppressor or an oncogene depending on the specific cellular context.

The analysis of PDE2A expression in relation to clinicopathological features, prognostic outcomes, genetic alterations, immunological functions, and drug sensitivity provides insights into its complex roles in cancer biology . This makes PDE2A a promising candidate as both a prognostic marker and a potential therapeutic target in multiple cancer types.

What techniques are most effective for quantifying PDE2A expression in tissue samples?

Several complementary techniques can be employed for accurate quantification of PDE2A expression in tissue samples, each with specific advantages:

• Quantitative Western Blot Analysis: Western blotting provides a reliable method for quantifying PDE2A protein levels. For accurate quantification, it is essential to use appropriate loading controls such as actin . The Li-Cor Odyssey system, which employs fluorescent secondary antibodies (e.g., anti-rabbit Alexa 680), offers a quantitative approach for measuring band intensity . This technique allows for the determination of relative PDE2A expression levels across different samples.

• Quantitative Real-Time PCR (qRT-PCR): For quantification of PDE2A mRNA levels, qRT-PCR provides a sensitive and specific method. This approach is particularly useful for comparing expression levels across different tissues or under various experimental conditions.

• RNA-Seq Analysis: For a comprehensive analysis of PDE2A expression across different tissues or cancer types, RNA-Seq data from repositories such as TCGA and GTEx can be utilized . These data are typically represented on a log2 (TPM + 1) scale, with TPM denoting transcripts per million .

• Proteomics Analysis: Proteomic approaches, such as those employed by the Clinical Proteomic Tumor Analysis Consortium (CPTAC), provide insights into PDE2A protein expression patterns across diverse tissue types .

• Image Analysis of Immunohistochemistry: Quantitative analysis of immunohistochemistry images allows for the assessment of both the intensity and distribution of PDE2A staining in tissue sections. This approach is particularly valuable for examining regional and cellular differences in PDE2A expression within complex tissues like the brain .

Each of these techniques offers unique insights into PDE2A expression, and a combination of approaches is often necessary for a comprehensive understanding of its expression patterns in normal and pathological conditions.

How can PDE2A antibodies be used to investigate neuronal signaling pathways?

PDE2A antibodies serve as powerful tools for investigating the role of this enzyme in neuronal signaling pathways. PDE2A functions as a dual-specificity phosphodiesterase that hydrolyzes both cAMP and cGMP, key second messengers involved in numerous signaling cascades . By utilizing PDE2A antibodies in various experimental approaches, researchers can gain insights into how this enzyme regulates neuronal communication:

• Colocalization Studies: Combining PDE2A antibodies with markers for specific neuronal populations or subcellular compartments allows for the identification of where PDE2A functions within neuronal circuits. The predominant localization of PDE2A in axons and nerve terminals suggests its involvement in presynaptic regulation . Immunofluorescence studies using PDE2A antibodies alongside synaptic markers can reveal its precise localization at synapses.

• Activity-Dependent Regulation: PDE2A antibodies can be used to investigate how neuronal activity modulates PDE2A expression or localization. This approach helps understand the dynamic regulation of cAMP/cGMP signaling in response to neuronal stimulation.

• Signaling Pathway Analysis: Through immunoprecipitation with PDE2A antibodies followed by mass spectrometry or co-immunoprecipitation with other proteins, researchers can identify PDE2A-interacting partners within signaling complexes. This helps map the signaling networks in which PDE2A participates.

• Circuit-Level Investigations: The specific distribution of PDE2A across different brain regions (such as cortex, hippocampus, and striatum) suggests its involvement in regulating specific neural circuits . PDE2A antibodies allow for the identification of these circuits and the investigation of how PDE2A modulation affects their function.

• Phosphorylation Studies: Combining PDE2A antibodies with phospho-specific antibodies enables the investigation of how post-translational modifications regulate PDE2A activity within neuronal signaling pathways.

These applications collectively contribute to our understanding of how PDE2A regulation of cAMP/cGMP impacts neuronal communication, synaptic plasticity, and ultimately, higher brain functions.

PDE2A antibodies have emerging applications in cancer research based on recent findings that PDE2A expression varies significantly across different cancer types and may serve as a novel biomarker . Several specific applications of PDE2A antibodies in cancer research include:

• Diagnostic and Prognostic Biomarker Development: PDE2A antibodies can be used in immunohistochemical analysis of tumor biopsies to assess protein expression levels. Given that PDE2A levels are notably lower in several cancer types (including adrenocortical carcinoma, acute myeloid leukemia, lower-grade glioma, ovarian cancer, testicular germ cell tumors, and uterine carcinosarcoma) compared to corresponding normal tissues , immunohistochemical staining patterns may provide valuable diagnostic or prognostic information.

• Tumor Microenvironment Analysis: PDE2A expression has been linked to immune cell infiltration and tumor microenvironment characteristics . PDE2A antibodies, in combination with markers for immune and stromal cells, can help analyze the relationship between PDE2A expression and the tumor immune microenvironment, potentially informing immunotherapeutic approaches.

• Identification of PDE2A-Associated Signaling Networks: Through techniques such as immunoprecipitation followed by mass spectrometry, PDE2A antibodies can help identify protein interaction networks specific to cancer cells. This information can reveal how altered PDE2A expression impacts signaling pathways that contribute to cancer progression.

• Therapeutic Target Validation: In cases where PDE2A is being investigated as a potential therapeutic target, antibodies are essential for confirming target engagement of PDE2A inhibitors and evaluating their effects on downstream signaling pathways in cancer cells.

• Monitoring Treatment Response: PDE2A antibodies could potentially be used to monitor changes in protein expression following treatment, serving as a pharmacodynamic marker of response to therapy, particularly for treatments that might affect cAMP/cGMP signaling pathways.

The comprehensive pan-cancer analysis of PDE2A expression, incorporating both transcriptomic data from TCGA and GTEx databases and proteomic data from the CPTAC database , provides a foundation for these applications. By correlating PDE2A expression with clinicopathological features, prognostic outcomes, genetic alterations, immunological functions, and drug sensitivity, researchers can develop more targeted approaches to cancer diagnosis and treatment.

What are common challenges in PDE2A immunodetection and how can they be overcome?

Researchers frequently encounter several challenges when working with PDE2A antibodies for immunodetection. Here are the most common issues and strategies to address them:

• Variable Signal Intensity Across Tissues: PDE2A expression varies significantly across different brain regions and tissues . The density of the PDE2A 100-kDa band appears to be less abundant in human striatum compared to other species , which can lead to inconsistent results when comparing across species or tissues.

  • Solution: Optimize antibody concentration for each specific tissue type and adjust exposure times accordingly. Consider using more sensitive detection systems for tissues with lower expression levels.

• Background Staining: Non-specific binding can obscure specific PDE2A signals, particularly in immunohistochemistry applications.

  • Solution: Implement more stringent blocking procedures using appropriate blocking agents (BSA, normal serum, or commercial blocking solutions). Optimize primary antibody dilution (between 1:20-1:200 for IHC as recommended ) and include appropriate negative controls (no primary antibody and, ideally, tissue from PDE2A knockout models).

• Antigen Masking in Fixed Tissues: Formalin fixation can mask epitopes and reduce antibody binding.

  • Solution: Implement effective antigen retrieval methods. For PDE2A antibody (55306-1-AP), using TE buffer at pH 9.0 is recommended, with citrate buffer at pH 6.0 as an alternative . Optimize the retrieval time and temperature for specific tissue types.

• Cross-Reactivity: Antibodies may recognize proteins with similar epitopes to PDE2A.

  • Solution: Validate antibody specificity through immunoadsorption tests where the antibody is preincubated with its immunogenic peptide . All specific bands should be abolished after this treatment. Additionally, Western blot analysis should show a single band at the expected molecular weight (approximately 100-106 kDa) .

• Inconsistent Results in Quantitative Analysis: Variations in sample preparation, antibody performance, and detection methods can lead to quantitative inconsistencies.

  • Solution: Implement standardized protocols for sample preparation, use consistent lots of antibody, include appropriate internal controls in each experiment, and employ quantitative detection systems like the Li-Cor Odyssey system for Western blot analysis.

• Limited Detection in Subcellular Compartments: PDE2A's preferential localization to axons and nerve terminals with weaker expression in cell bodies can make detection challenging in certain experimental setups.

  • Solution: Use subcellular fractionation techniques to enrich for specific compartments before Western blot analysis, or employ high-resolution microscopy techniques for immunofluorescence studies to better visualize compartment-specific localization.

How should researchers interpret discrepancies between mRNA and protein expression data for PDE2A?

Discrepancies between PDE2A mRNA and protein expression are commonly observed and can provide valuable insights into post-transcriptional regulation mechanisms. Here's how researchers should approach and interpret such discrepancies:

• Differential Subcellular Localization: Studies have shown that while PDE2A mRNA is expressed in the cell bodies of neurons (such as pyramidal cells in cortex and hippocampus), the protein is predominantly localized to axons and nerve terminals, with markedly weaker expression in cell bodies . This pattern suggests active transport of PDE2A protein from the soma to axonal compartments.

  • Interpretation: Such discrepancies indicate compartment-specific regulation of PDE2A and highlight the importance of examining both mRNA localization (through in situ hybridization) and protein distribution (through immunohistochemistry) to understand the full biological context.

• Temporal Dynamics: Differences between mRNA and protein levels may reflect temporal dynamics in gene expression and protein synthesis/degradation.

  • Interpretation: Time-course studies measuring both mRNA (via qRT-PCR) and protein (via Western blot) can help elucidate these dynamics. Consider that mRNA changes often precede corresponding changes in protein levels.

• Post-Transcriptional Regulation: Discrepancies may reflect post-transcriptional regulatory mechanisms such as microRNA-mediated repression, RNA binding proteins, or alterations in mRNA stability.

  • Interpretation: Complementary studies investigating these regulatory mechanisms may be necessary to explain consistent discrepancies between mRNA and protein levels.

• Technical Variations: Different sensitivities and specificities of techniques used to measure mRNA (e.g., qRT-PCR, RNA-Seq) versus protein (e.g., Western blot, immunohistochemistry) can contribute to apparent discrepancies.

  • Interpretation: Employ multiple complementary techniques to measure both mRNA and protein. For instance, combine RNA-Seq with quantitative proteomics to obtain comprehensive expression profiles.

• Disease-Specific Alterations: In pathological conditions such as cancer, the relationship between mRNA and protein expression may be altered due to dysregulated post-transcriptional mechanisms.

  • Interpretation: The finding that PDE2A levels are lower in several cancer types should be validated at both mRNA and protein levels to determine whether the downregulation occurs at the transcriptional or post-transcriptional level.

A methodical approach to investigating these discrepancies includes parallel analysis of mRNA and protein in the same samples, subcellular fractionation studies, and temporal profiling following experimental manipulations. These approaches collectively provide a more comprehensive understanding of PDE2A regulation.

How can researchers effectively compare PDE2A expression data across different experimental platforms and species?

Comparing PDE2A expression data across different experimental platforms and species presents significant challenges due to methodological variations, species-specific differences, and diverse quantification approaches. Here are strategies for effective cross-platform and cross-species comparisons:

• Standardized Reference Samples: Include common reference samples across different experimental platforms.

  • Implementation: Process a set of standardized samples (e.g., specific brain regions from a common source) across all platforms being compared. These samples serve as calibration standards to normalize data across platforms.

• Cross-Platform Normalization Methods: Employ statistical normalization techniques specifically designed for cross-platform comparisons.

  • Implementation: Techniques such as quantile normalization, ComBat for batch effect correction, or more advanced machine learning approaches can help integrate data from different platforms. For instance, when combining RNA-Seq data from TCGA with proteomics data from CPTAC , appropriate normalization is essential.

• Orthologous Gene/Protein Identification: Carefully identify true orthologs when comparing across species.

  • Implementation: Use established databases like HomoloGene or OrthoDB to identify orthologous PDE2A genes/proteins across species. Consider sequence conservation, especially in the regions targeted by antibodies or primers.

• Conserved Domain Analysis: Focus comparisons on highly conserved functional domains of PDE2A.

  • Implementation: When examining protein expression or function, prioritize analyses of conserved catalytic domains that are more likely to maintain similar functions across species.

• Relative Expression Patterns: Compare relative expression patterns rather than absolute values.

  • Implementation: Instead of directly comparing raw expression values, analyze relative expression patterns across different tissues or conditions within each species, then compare these patterns between species. Studies have shown that the pattern of PDE2A immunoreactivity is consistent across rats, mice, cynomolgus monkeys, dogs, and humans , suggesting conservation of expression patterns despite potential differences in absolute levels.

• Complementary Validation Approaches: Validate findings using multiple independent techniques.

  • Implementation: If a specific PDE2A expression pattern is observed using one platform (e.g., RNA-Seq), validate it using independent methods (e.g., qRT-PCR, Western blot, immunohistochemistry) before making cross-species comparisons.

• Metadata Integration: Carefully document and consider experimental variables.

  • Implementation: Maintain comprehensive metadata regarding experimental conditions, sample processing, technical specifications of platforms, and analysis parameters. These factors can significantly impact results and should be considered when comparing across studies.