PDE11A Antibody, FITC conjugated

What is PDE11A and why is it important in cellular signaling research?

PDE11A (Phosphodiesterase 11A) is a critical enzyme that regulates intracellular concentrations of cyclic nucleotides cAMP and cGMP. It catalyzes the hydrolysis of both cAMP and cGMP to 5'-AMP and 5'-GMP, respectively, making it a dual-substrate phosphodiesterase. This dual functionality allows PDE11A to potentially regulate both signaling pathways under physiological conditions, making it a significant target for research in signal transduction mechanisms. The enzyme contains GAF domains and an N-terminal flanking sequence with consensus sequences for PKA and protein kinase G phosphorylation, contributing to its regulatory complexity .

What are the known isoforms of PDE11A and their tissue distribution?

There are multiple isoforms of PDE11A (PDE11A1-PDE11A4) with distinct tissue expression patterns. Isoform 1 is present in prostate, pituitary, heart, and liver, but notably absent in testis and penis. This tissue-specific distribution suggests that weak inhibition by medications like Tadalafil may not be relevant in certain tissues. Isoform 2 appears to be primarily expressed in testis. This differential expression pattern is important for researchers studying tissue-specific functions and when selecting appropriate experimental models .

What applications are supported by FITC-conjugated PDE11A antibodies?

FITC-conjugated PDE11A antibodies support multiple research applications including confocal microscopy (CM), enzyme-linked immunosorbent assay (ELISA), immunocytochemistry (ICC), immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation (IP), and Western blotting (WB). The FITC conjugation provides direct fluorescent detection capability, eliminating the need for secondary antibody incubation in fluorescence-based applications. This versatility makes these antibodies valuable tools for multiple experimental approaches in studying PDE11A localization and function .

What species reactivity should be considered when selecting a PDE11A antibody?

When selecting a PDE11A antibody, researchers should carefully consider species reactivity based on their experimental model. Commercial FITC-conjugated PDE11A antibodies show varying reactivity profiles. Some antibodies react with human, mouse, and rat PDE11A, while others may only react with human PDE11A. This difference in reactivity is critical for cross-species studies and when translating findings between animal models and human samples. Always verify the specific reactivity of your antibody before designing cross-species experiments .

What is the recommended storage condition for maintaining FITC-conjugated PDE11A antibody activity?

For optimal preservation of FITC-conjugated PDE11A antibody activity, storage at -20°C is recommended for long-term stability. The antibodies are typically supplied in a stabilization buffer containing components like glycerol (50%) and PBS (pH 7.4) with preservatives such as Proclin 300 (0.03%). These conditions help maintain both antibody integrity and FITC fluorescence. Repeated freeze-thaw cycles should be avoided as they can degrade both the antibody and the fluorophore, reducing detection sensitivity in experimental applications .

How can PDE11A antibodies be utilized in studies of genetic variants associated with adrenocortical tumors?

For investigating PDE11A genetic variants associated with adrenocortical tumors, researchers can design experiments combining FITC-conjugated PDE11A antibodies with specific genetic analysis. Studies have shown that PDE11A variants are more frequent in patients with ACTH-independent macronodular adrenal hyperplasia (AIMAH) (28% compared to 7.2% in controls). When studying these variants, researchers can use the antibodies in immunofluorescence assays to examine protein localization differences between wild-type and variant forms. Additionally, these antibodies can be employed in co-localization studies with PKA signaling components to understand how PDE11A variants might alter cAMP signaling pathways. For functional studies, researchers should consider combining antibody-based detection with FRET-based cAMP sensors to monitor real-time changes in cAMP levels in cells expressing different PDE11A variants .

What are the considerations for using PDE11A antibodies in FRET experiments studying cAMP dynamics?

When incorporating PDE11A antibodies in FRET experiments studying cAMP dynamics, several factors must be considered. Researchers should first establish appropriate controls with wild-type PDE11A expression to establish baseline cAMP regulation. Previous research has utilized Epac1-camps as a cAMP sensor with variants of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as FRET donor and acceptor, respectively. When co-transfecting cells with FRET sensors and plasmids expressing either wild-type or variant forms of PDE11A, the recommended protocol involves using 0.4 μg of FRET sensor DNA and 24 pmol of plasmid DNA. Experiments should be performed 48 hours after transfection, and measurements should include baseline readings followed by stimulation with agents like forskolin to observe dynamic changes in cAMP levels. The FITC-conjugated antibodies can be used in parallel experiments to confirm expression and localization of the transfected PDE11A constructs .

How do disease-associated PDE11A variants differ functionally from wild-type, and how can antibodies help characterize these differences?

Disease-associated PDE11A variants (such as D609N and M878V found in AIMAH patients) show functional differences from wild-type PDE11A that can be characterized using antibodies. These variants demonstrate altered enzymatic activity, particularly in their capacity to regulate cAMP levels. In experimental settings, cells transfected with these variant forms show higher cAMP levels after forskolin stimulation compared to wild-type PDE11A, suggesting reduced catalytic function. FITC-conjugated PDE11A antibodies can be used to confirm equal expression levels of wild-type and variant forms before functional assays, ensuring that observed differences are due to functional alterations rather than expression variability. Additionally, these antibodies can be used to investigate potential differences in subcellular localization between wild-type and variant forms, which might contribute to the observed functional differences .

What are the optimal dilution ratios for different experimental applications?

The optimal dilution ratios for FITC-conjugated PDE11A antibodies vary by application. For Western blotting, a dilution of 1:10,000 is typically recommended, while immunofluorescence, immunohistochemistry, immunocytochemistry, and immunoprecipitation typically require more concentrated antibody at approximately 1:250. For confocal microscopy, a dilution of 1:250 is suggested, and ELISA applications generally use a 1:500 dilution. These recommendations serve as starting points, and researchers should perform titration experiments to determine the optimal concentration for their specific experimental conditions, tissue types, and detection systems. Over-dilution may result in weak signals, while insufficient dilution could lead to high background or non-specific binding .

How can researchers validate the specificity of PDE11A antibodies in their experimental systems?

To validate the specificity of PDE11A antibodies, researchers should implement multiple control strategies. Negative controls should include samples known to lack PDE11A expression or tissues from PDE11A knockout models if available. Competitive blocking experiments using the immunizing peptide can confirm binding specificity. Additionally, researchers should verify that the antibody detects the expected molecular weight bands in Western blot applications (the specific weight will depend on which isoform is being detected). Cross-reactivity testing is also important; high-quality PDE11A antibodies should not cross-react with other PDE family members. For FITC-conjugated antibodies specifically, researchers should include controls to account for potential autofluorescence in their experimental system and validate fluorescence specificity with parallel experiments using unconjugated primary antibodies detected with FITC-labeled secondary antibodies .

What approaches can be used to quantify PDE11A expression levels using FITC-conjugated antibodies?

For quantifying PDE11A expression levels using FITC-conjugated antibodies, researchers can employ several approaches depending on the experimental context. Flow cytometry offers a high-throughput method for quantifying expression in cell populations, with mean fluorescence intensity correlating to protein expression levels. For tissue sections or cultured cells, quantitative image analysis of immunofluorescence signals can be performed using software that measures integrated density or mean fluorescence intensity within defined regions of interest. Western blot analysis with FITC detection can also be used for semi-quantitative assessment, though researchers should include standard curves with recombinant PDE11A protein for more accurate quantification. In all approaches, it's crucial to include calibration standards and normalize to appropriate housekeeping proteins or total protein content. Additionally, when comparing expression between experimental groups, all samples should be processed simultaneously with identical antibody concentrations, incubation times, and imaging parameters to ensure valid comparisons .

How can FITC-conjugated PDE11A antibodies be integrated with other fluorescent markers for co-localization studies?

When designing co-localization studies with FITC-conjugated PDE11A antibodies, researchers must carefully select compatible fluorophores to avoid spectral overlap. FITC emits in the green spectrum (peak emission ~520 nm), so complementary fluorophores should emit in distinctly different spectral ranges, such as red (e.g., Texas Red, Cy3) or far-red (e.g., Cy5, Alexa 647) channels. For subcellular localization studies, researchers can combine FITC-PDE11A antibodies with organelle-specific markers such as DAPI (nucleus), MitoTracker (mitochondria), or antibodies against organelle-specific proteins conjugated to compatible fluorophores. When conducting co-localization analysis, appropriate controls should include single-stained samples to establish bleed-through parameters and unstained samples to determine autofluorescence levels. Quantitative co-localization analysis can be performed using software that calculates Pearson's correlation coefficient or Manders' overlap coefficient. This approach is particularly valuable for determining whether PDE11A co-localizes with components of cAMP signaling pathways or whether disease-associated variants show altered localization patterns .

How does PDE11A interact with other components of cAMP and cGMP signaling pathways?

PDE11A functions as a dual-specificity phosphodiesterase that hydrolyzes both cAMP and cGMP with similar efficiency (Km values of 0.52 μM and 1.04 μM, respectively). In signaling pathway studies, researchers can use FITC-conjugated PDE11A antibodies in combination with antibodies against other pathway components to map interaction networks. PDE11A contains GAF domains that may be involved in protein-protein interactions or allosteric regulation. To study these interactions, immunoprecipitation with PDE11A antibodies followed by mass spectrometry can identify binding partners. Additionally, PDE11A activity is sensitive to inhibitors like IBMX (IC50 49.8 μM), zaprinast (IC50 12.0 μM), and dipyridamole (IC50 0.37 μM), which provides tools for pharmacological manipulation during signaling studies. When designing such experiments, researchers should consider the tissue-specific expression of different PDE11A isoforms and their potential distinct roles in different signaling contexts .

What methodological approaches can be used to study PDE11A in relation to disease pathogenesis?

To investigate PDE11A's role in disease pathogenesis, researchers can employ multiple complementary approaches using FITC-conjugated antibodies. For genetic studies, researchers can collect patient samples with known PDE11A variants (such as those linked to adrenocortical tumors, Carney complex, or testicular tumors) and use immunofluorescence to assess expression patterns and levels compared to controls. Functional studies can incorporate FRET-based cAMP assays as described earlier to determine how disease-associated variants alter cAMP regulation. For translational research, tissue microarrays of patient samples can be analyzed using the antibodies to correlate PDE11A expression with clinical parameters. Cell models expressing disease-associated variants can be developed and characterized using the antibodies in combination with functional assays measuring proliferation, hormone production, or other relevant phenotypes. When studying PDE11A mutations in the catalytic domain (such as D609N and M878V), researchers should consider how these might affect interaction with inhibitors, which could have implications for therapeutic approaches .