PDE1A Antibody, HRP conjugated

What is PDE1A and why is it significant for research?

PDE1A (Phosphodiesterase 1A) is a calcium/calmodulin-dependent cyclic nucleotide phosphodiesterase with dual specificity for the second messengers cGMP and cAMP, which serve as key regulators in numerous important physiological processes . It demonstrates higher efficiency with cGMP compared to cAMP, contributing to the precise regulation of these signaling molecules . PDE1A plays a crucial role in maintaining cellular homeostasis by hydrolyzing cAMP to 5'AMP, effectively downregulating cAMP signaling pathways that respond to external stimuli . The enzyme's activity is notably modulated by calcium ions and calmodulin, highlighting its integration within calcium-mediated signaling networks that control diverse cellular functions .

What are the structural characteristics of PDE1A protein?

PDE1A belongs to the cyclic nucleotide phosphodiesterase family, specifically the PDE1 subfamily, with a characteristic dependency on calcium and calmodulin for activation . The protein demonstrates remarkable evolutionary conservation, with human PDE1A sharing 94% homology with its bovine counterpart, differing only in two short unique regions . This high degree of conservation suggests fundamental importance to cellular function across species . PDE1A exists in multiple variants including PDE1A1, PDE1A3, and PDE1A5, which may exhibit tissue-specific expression patterns and subtle functional differences . The protein is predominantly located in the cytosol and contains functional domains for catalytic activity, calmodulin binding, and regulatory control .

What are the validated applications for PDE1A antibodies?

PDE1A antibodies have been validated for multiple experimental applications including western blotting (WB), immunocytochemistry/immunofluorescence (ICC/IF), immunoprecipitation (IP), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) . For western blotting, the antibodies reliably detect the predicted 61 kDa band corresponding to PDE1A in various human cell lines including H1299 and HeLa . In immunofluorescence applications, these antibodies effectively localize PDE1A protein in paraformaldehyde-fixed cells with clear differentiation from nuclear staining . The cross-reactivity profile varies by manufacturer, with some antibodies specifically designed to detect PDE1A across human, mouse, and rat samples, while others are optimized for human samples only .

What are the optimal dilution ratios for different applications of HRP-conjugated PDE1A antibodies?

The optimal dilution ratios for HRP-conjugated PDE1A antibodies vary significantly depending on the specific application, sample type, and detection method. For western blotting, a dilution range of 1:500-1:1,000 typically provides optimal signal-to-noise ratio with minimal background . For immunohistochemistry and immunocytochemistry applications, more concentrated solutions at approximately 1:200 dilution are generally recommended to achieve sufficient binding and signal intensity . For ELISA applications, higher dilutions between 1:5,000-1:10,000 often yield the best results due to the highly sensitive nature of the assay format . It is crucial to perform dilution optimization experiments for each new lot of antibody, as manufacturing variations can affect optimal working concentrations.

How should researchers validate the specificity of PDE1A antibodies?

Validating PDE1A antibody specificity requires a multi-faceted approach combining positive and negative controls. Researchers should first conduct western blot analysis using tissue or cell types known to express PDE1A (such as brain, kidney, testes, or heart) to confirm detection of the expected 61 kDa band . Parallel experiments should include negative controls either using tissues from PDE1A knockout models or cells where PDE1A expression has been silenced through RNAi techniques . Cross-reactivity assessment is essential, particularly against other PDE family members, to ensure the antibody specifically recognizes PDE1A without detecting related proteins . For additional validation, researchers should perform peptide competition assays where pre-incubation of the antibody with the immunizing peptide should abolish specific binding in subsequent experiments .

What are common sources of background when using HRP-conjugated PDE1A antibodies?

Common sources of background when using HRP-conjugated PDE1A antibodies include endogenous peroxidase activity in tissue samples, non-specific binding to highly charged cellular components, and excessive antibody concentration . Endogenous peroxidase activity, particularly in tissues rich in peroxidases like liver and kidney, can be mitigated by incorporating a peroxidase quenching step using hydrogen peroxide solution (0.3-3% H₂O₂) prior to antibody incubation . Non-specific binding can be reduced by optimizing blocking solutions, with 5% BSA or 5% non-fat dry milk in TBS-T often providing improved results compared to standard blocking reagents . Excessive antibody concentration frequently causes high background, necessitating careful titration experiments to determine the minimum concentration that yields specific signal . Additionally, insufficient washing between steps can lead to residual unbound antibody contributing to background signals.

How can researchers optimize signal detection when using HRP-conjugated PDE1A antibodies?

Signal optimization with HRP-conjugated PDE1A antibodies requires attention to several experimental parameters. First, researchers should select an appropriate substrate system based on the required sensitivity, with enhanced chemiluminescence (ECL) substrates offering high sensitivity for western blotting and IHC applications . Fresh preparation of all reagents, particularly substrate solutions, ensures maximum enzymatic activity and signal generation . Incubation time adjustments can significantly impact signal quality, with longer primary antibody incubations (overnight at 4°C) often improving specific binding while minimizing background . Temperature modulation during incubation steps can enhance antibody-antigen interaction kinetics, with room temperature incubations typically producing faster but potentially less specific results compared to 4°C incubations . For weak signals, signal amplification systems such as tyramide signal amplification (TSA) can be incorporated to enhance detection without increasing background .

What strategies can help preserve the activity of HRP-conjugated antibodies?

Preserving HRP-conjugated antibody activity requires careful attention to storage and handling procedures. These antibodies should be stored at -20°C for long-term preservation, with aliquoting strongly recommended to avoid repeated freeze-thaw cycles that can denature both the antibody and the conjugated enzyme . When working with the antibody, keeping it cold (on ice or at 4°C) minimizes degradation during experimental procedures . Addition of stabilizing proteins such as BSA (0.1-1%) and preservatives like sodium azide (0.02-0.05%) to storage buffers can extend shelf-life, though it's important to note that sodium azide inhibits HRP activity and must be removed before use through dialysis or dilution . Protection from light is essential for fluorescently-tagged HRP conjugates to prevent photobleaching . Researchers should also monitor solution pH, as extreme pH conditions can permanently damage HRP activity, with optimal preservation occurring in slightly acidic to neutral pH ranges (pH 6.0-7.5) .

How is PDE1A antibody being used to study protein-protein interactions?

PDE1A antibody has emerged as a valuable tool for investigating protein-protein interactions through co-immunoprecipitation, proximity ligation assays, and pull-down experiments . Recent research has demonstrated that PDE1A physically interacts with YTHDF2, a crucial finding in elucidating the molecular mechanisms underlying non-small cell lung cancer progression . For successful co-immunoprecipitation experiments, researchers typically use non-conjugated PDE1A antibodies bound to protein A/G beads to pull down PDE1A and its interacting partners, followed by western blot analysis to identify associated proteins . The specificity of these interactions can be verified through reverse co-immunoprecipitation experiments using antibodies against the putative binding partners . For studying dynamic interactions in intact cells, proximity ligation assays employing PDE1A antibodies in combination with antibodies against potential interactors enable visualization of protein complexes in their native cellular context with spatial resolution .

What role does PDE1A play in pathological conditions and how are antibodies facilitating this research?

PDE1A antibodies are instrumental in uncovering the roles of this enzyme in various pathological conditions, including cancer and cardiovascular disorders . In cancer research, PDE1A antibodies have facilitated the discovery of its involvement in non-small cell lung cancer (NSCLC) progression through interaction with YTHDF2, providing new insights into potential therapeutic targets . Immunohistochemical analysis using PDE1A antibodies allows researchers to compare expression levels between normal and diseased tissues, revealing alterations associated with pathological states . For mechanistic studies, researchers employ PDE1A antibodies in combination with pharmacological inhibitors to dissect the signaling pathways regulated by this enzyme under normal and pathological conditions . The development of phospho-specific PDE1A antibodies has enabled examination of the enzyme's regulation through post-translational modifications in disease contexts, adding another layer of complexity to our understanding of PDE1A biology .

How can multiplexed imaging be achieved using PDE1A antibodies alongside other markers?

Multiplexed imaging using PDE1A antibodies alongside other cellular markers requires strategic selection of compatible detection systems and careful experimental design . For fluorescence-based multiplexing, researchers can use PDE1A-HRP conjugated antibodies with tyramide signal amplification systems that deposit fluorescent tyramide, which remains covalently bound during subsequent antibody stripping procedures . This approach allows sequential detection of multiple proteins using primary antibodies from the same species without cross-reactivity . Alternative strategies include using PDE1A antibodies from different host species (rabbit polyclonal versus mouse monoclonal) in combination with spectrally distinct fluorophore-conjugated secondary antibodies . For chromogenic multiplexing in tissue sections, HRP-conjugated PDE1A antibodies can be used with one chromogen, followed by antibody stripping and sequential detection of additional markers using alkaline phosphatase-conjugated antibodies with contrasting chromogens .

How might single-cell analysis benefit from advanced PDE1A antibody applications?

Single-cell analysis represents a frontier where advanced PDE1A antibody applications could significantly enhance our understanding of cellular heterogeneity in phosphodiesterase signaling . HRP-conjugated PDE1A antibodies, when adapted for techniques like imaging mass cytometry or cyclic immunofluorescence, could enable quantitative assessment of PDE1A expression and localization at the single-cell level within complex tissues . This approach would reveal population-specific variations in PDE1A expression that are obscured in bulk tissue analyses . By combining PDE1A detection with markers for cell type, cell cycle stage, and activation state, researchers could construct comprehensive maps of PDE1A regulation across diverse cellular contexts . Emerging technologies like in situ sequencing could be paired with PDE1A immunodetection to correlate protein expression with transcriptional profiles at single-cell resolution, providing unprecedented insights into the regulatory networks governing PDE1A function in both normal physiology and disease states .

What are the emerging applications of PDE1A antibodies in translational research?

Emerging applications of PDE1A antibodies in translational research span diagnostic, prognostic, and therapeutic domains . In oncology, PDE1A antibody-based assays are being developed to assess PDE1A expression as a potential biomarker for cancer progression and treatment response, particularly in non-small cell lung cancer where PDE1A appears to play a significant role . Immunohistochemical analysis of patient tissue samples using PDE1A antibodies is helping to stratify patients based on expression patterns, potentially guiding personalized treatment approaches . In drug discovery pipelines, PDE1A antibodies are facilitating high-throughput screening assays to identify novel PDE1A inhibitors with improved selectivity profiles . Additionally, conjugation of PDE1A antibodies to nanoparticles or drug-delivery systems is being explored for targeted therapeutic approaches that could modulate PDE1A activity specifically in diseased tissues while sparing normal cells .

How can computational approaches enhance the utility of PDE1A antibody research data?

Computational approaches are increasingly essential for maximizing the value of PDE1A antibody research data across multiple experimental contexts . Machine learning algorithms applied to immunohistochemistry images can quantify subtle variations in PDE1A expression patterns across different tissue regions and correlate these with clinical outcomes, extracting information that might escape visual inspection . Network analysis integrating PDE1A protein interaction data from immunoprecipitation studies with transcriptomic and proteomic datasets can reveal functional modules and signaling hubs where PDE1A plays central roles . Molecular dynamics simulations informed by structural data can predict how antibody binding might affect PDE1A function, guiding the development of antibodies with desired modulatory properties . For high-dimensional cytometry data, dimensionality reduction techniques and clustering algorithms can identify cell populations with distinct PDE1A expression profiles, establishing correlations with cellular phenotypes and functional states .