Pde5a Antibody

What is PDE5A and why is it important in vascular research?

PDE5A (phosphodiesterase 5A) is an enzyme that plays a critical role in hydrolyzing cyclic guanosine monophosphate (cGMP) to its inactive form (5'-GMP). It specifically regulates nitric-oxide-generated cGMP, making it central to vascular tone regulation. While traditionally associated with smooth muscle cells, recent research demonstrates that PDE5A is also expressed in endothelial cells where it forms a feedback loop with nitric oxide synthase (NOS3). This endothelial PDE5A localization is primarily at or near caveolae, allowing for critical spatial regulation of cGMP signaling pathways . Inhibition of endothelial PDE5A has been shown to improve endothelial function, offering therapeutic potential for vascular conditions such as pulmonary hypertension.

How do I select the appropriate PDE5A antibody for my specific research application?

When selecting a PDE5A antibody, consider:

• Experimental application: Different applications require antibodies with specific validation profiles. For detecting PDE5A in Western blots, antibodies validated for WB are essential. For localization studies, choose antibodies validated for ICC/IF or IHC-P .

• Species reactivity: Ensure the antibody recognizes PDE5A in your model organism. Common reactivities include human, mouse, and rat PDE5A, but cross-reactivity varies between antibodies .

• Epitope location: Antibodies targeting different epitopes within PDE5A may yield different results. Some antibodies target regions within amino acids 300-450, while others target different domains . This is particularly important when studying specific PDE5A isoforms.

• Published validation: Prioritize antibodies cited in peer-reviewed publications for your specific application and experimental context .

• Clone type: Consider whether polyclonal antibodies (offering multiple epitope recognition) or monoclonal antibodies (offering high specificity) better suit your experimental needs.

What is the optimal protocol for using PDE5A antibodies in Western blot applications?

For optimal Western blot detection of PDE5A:

• Sample preparation:

  • Lyse tissues or cells in RIPA buffer containing protease inhibitors

  • PDE5A is primarily detected as a ~100 kDa band in bovine and human tissues

  • Include phosphatase inhibitors if studying PDE5A phosphorylation states

• Gel electrophoresis:

  • Use 8% SDS-PAGE gels for optimal separation of the ~100 kDa PDE5A protein

  • Load 20-40 μg of total protein per lane

• Transfer and blocking:

  • Transfer proteins to PVDF membrane (preferred over nitrocellulose for PDE5A)

  • Block in 5% non-fat milk in TBST for 1 hour at room temperature

• Antibody incubation:

  • Dilute primary PDE5A antibody at 1:1000 in 5% BSA in TBST

  • Incubate overnight at 4°C with gentle agitation

  • Wash extensively with TBST (4 × 10 minutes)

  • Incubate with appropriate HRP-conjugated secondary antibody at 1:5000 for 1 hour

• Detection:

  • Use enhanced chemiluminescence (ECL) detection

  • PDE5A typically appears as a distinct band at approximately 100 kDa

  • Verify specificity using positive controls (lung or platelets express high levels of PDE5A)

How can I effectively use PDE5A antibodies for immunohistochemistry and immunofluorescence studies?

For optimal IHC-P and IF detection of PDE5A:

• Tissue fixation and preparation:

  • Fix tissues in 4% paraformaldehyde

  • For paraffin sections: 5 μm thickness is optimal

  • For frozen sections: 8-10 μm thickness works well

• Antigen retrieval:

  • Critical for PDE5A detection in paraffin-embedded tissues

  • Use citrate buffer (pH 6.0) and heat-induced epitope retrieval

  • Boil sections for 15-20 minutes, followed by cooling to room temperature

• Blocking and permeabilization:

  • Block with 5-10% normal serum from the species of secondary antibody

  • Add 0.1-0.3% Triton X-100 for permeabilization in IF staining

  • Block endogenous peroxidase with 3% H₂O₂ for IHC-P

• Antibody incubation:

  • Use PDE5A antibody at 1:200 dilution for IHC-P applications

  • For IF, a 1:100-1:200 dilution range is typically effective

  • Incubate at 4°C overnight in a humidified chamber

• Detection and visualization:

  • For IHC-P: Use appropriate detection system (e.g., HRP-polymer and DAB)

  • For IF: Use fluorophore-conjugated secondary antibodies

  • Include DAPI nuclear counterstain for IF

• Expected staining patterns:

  • Cytoplasmic positivity in various cell types

  • Moderate cytoplasmic staining in pancreatic intercalated ducts

  • Weak to moderate staining in duodenal glandular cells

How can I investigate the spatial relationship between PDE5A and NOS3 in endothelial cells?

To study the PDE5A-NOS3 spatial relationship:

• Co-immunoprecipitation approach:

  • Immunoprecipitate PDE5A from endothelial cell lysates

  • Probe for NOS3 co-precipitation by Western blot

  • Reciprocal IP (pull down NOS3, probe for PDE5A) to confirm interaction

• Proximity ligation assay (PLA):

  • Use primary antibodies against PDE5A and NOS3 from different species

  • Apply species-specific PLA probes and perform ligation and amplification

  • Fluorescent dots indicate proteins in close proximity (<40 nm)

• Confocal microscopy co-localization:

  • Perform double immunofluorescence for PDE5A and NOS3

  • Analyze co-localization using appropriate software (e.g., ImageJ with JACoP plugin)

  • Quantify using Pearson's or Mander's coefficients

• Subcellular fractionation approach:

  • Isolate caveolin-rich lipid raft fractions as described in published protocols

  • Analyze PDE5A and NOS3 distribution by Western blot

  • Expect co-expression of caveolin-1, PDE5A, and NOS3 in the same fractions

• Immunogold electron microscopy:

  • Use 6 nm immunogold particles for PDE5A detection

  • Target PDE5A within subendothelial invaginations

  • Quantify distribution relative to cellular structures

Research has demonstrated that PDE5A is heavily concentrated within subendothelial invaginations, with immunogold particles showing concentration of PDE5A in these regions .

How can I establish an enzymatic activity assay for PDE5A to test potential inhibitors?

Two validated approaches for PDE5A enzymatic activity assays:

Method 1: Label-free LC/MS-based enzymatic activity assay

• Reaction setup:

  • 150 μL reaction volume containing:

    • 50 nM PDE5A enzyme

    • Test compounds at various concentrations

    • 10 μM cGMP substrate

  • Pre-incubate PDE5A with compounds for 30 min at room temperature

  • Add cGMP and incubate for 15 min at room temperature

• Sample preparation:

  • Filter the reaction mixture through an ultrafiltration membrane to remove protein

  • Analyze the filtrate by LC/MS

• Analysis:

  • Detect and quantify GMP release

• IC₅₀ determination:

  • Test compounds at multiple concentrations

  • Plot concentration vs. inhibition

  • Calculate IC₅₀ values using non-linear regression

  • Validated IC₅₀ values: sildenafil (78.72 ± 1.7 nM) and vardenafil (1.47 ± 0.02 nM)

Method 2: Fluorescent-labeled substrate-based method

• Alternative approach using fluorescently labeled cGMP substrate

• Provides comparable results to the LC/MS method

• Measured IC₅₀ values: sildenafil (100.7 ± 4.18 nM) and vardenafil (3.98 ± 0.08 nM)

What approaches can be used to study PDE5A-mediated regulation of NOS3 activity?

To investigate PDE5A regulation of NOS3:

• PDE5A overexpression studies:

  • Transfect endothelial cells with adenoviral PDE5A vector (AdPDE5A)

  • Monitor effects on:

    • NOS3 phosphorylation at S1179 (decreases by ~50%)

    • Total NOS3 expression (increases)

    • Arginine-to-citrulline conversion (decreases by ~50%)

    • NO accumulation in culture medium (decreases by ~25%)

• PDE5A inhibition studies:

  • Treat endothelial cells with PDE5A inhibitors (e.g., sildenafil, tadalafil)

  • Typical treatment: 1 μM for 48 hours

  • Measure increased NO production (~20% increase)

  • Assess NOS3 activity via DAF2 fluorescence

• PKG1 manipulation:

  • Overexpress PKG1 using adenoviral vectors

  • Silence PKG1 using siRNA

  • Measure effects on NOS3 phosphorylation and activity

  • PKG1 overexpression increases NOS3 activity by ~35%

• Flow-dependent vasodilation experiments:

  • Transfect pulmonary vascular rings with AdPDE5A

  • Measure flow-mediated vasodilation

  • Compare to controls (Adβgal-transfected rings)

  • Use L-NAME as positive control for NOS inhibition

How can PDE5A antibodies be used to study pulmonary hypertension pathophysiology?

PDE5A is significantly upregulated in pulmonary hypertension, making it a valuable biomarker and therapeutic target. To study its role:

• Patient-derived samples:

  • Isolate pulmonary endothelial cells from patients with pulmonary hypertension

  • Compare PDE5A expression with cells from healthy donors

  • Expect significant increase in PDE5A expression and activity in PH samples

• PDE5A inhibitor studies:

  • Treat pulmonary endothelial cells from PH patients with PDE5A inhibitors

  • Measure cGMP-dependent PDE activity

  • Expect >60% inhibition of total cGMP-dependent PDE activity with sildenafil or tadalafil (1 μM)

• Immunohistochemical analysis:

  • Perform PDE5A immunostaining on lung sections from PH patients

  • Compare with healthy lung tissue

  • Quantify expression differences in:

    • Pulmonary arterial smooth muscle cells

    • Pulmonary endothelial cells

    • Potential remodeled areas

• Functional studies:

  • Measure endothelium-dependent vasodilation in pulmonary arteries

  • Compare responses with and without PDE5A inhibition

  • Assess flow-mediated dilation as a marker of endothelial function

How can I effectively screen for novel PDE5A inhibitors using antibody-based techniques?

A comprehensive approach to PDE5A inhibitor screening:

• Virtual screening and molecular docking :

  • Dock compounds into the catalytic site of PDE5A

  • Select candidates based on affinity scores and visual inspection

  • Example: From 1,427 compounds screened, 10 were selected for further testing

• In vitro inhibition assays:

  • Use LC/MS-based enzymatic activity assay

  • Test compounds at 20 μM initial concentration

  • Select active compounds showing >50% inhibition for IC₅₀ determination

  • Example: Proanthocyanidins showed 91% inhibition at 20 μM with IC₅₀ of 870 ± 0.02 nM

• Binding mode analysis:

  • Compare binding modes of novel inhibitors with established ones (e.g., sildenafil)

  • Analyze hydrogen bond formation with key residues (e.g., Gln817, Gln789)

  • Examine occupation of active site and extensions to adjacent regions

  • Example: Proanthocyanidins formed additional hydrogen bonds with Gln789 compared to sildenafil

• Structure-activity relationship studies:

  • Compare inhibitory activity of structurally related compounds

  • Identify critical functional groups for inhibition

  • Guide rational design of improved inhibitors

What are the key considerations for validating PDE5A antibody specificity?

To ensure the validity of your PDE5A antibody results:

• Positive control tissues/cells:

  • Lung tissue (high PDE5A expression)

  • Platelets (known PDE5A expression)

  • Vascular smooth muscle cells

  • Pancreatic intercalated ducts

  • Duodenal glandular cells

• Negative controls:

  • Omission of primary antibody

  • Isotype control antibody

  • Pre-absorption with immunizing peptide

  • PDE5A knockout tissues (if available)

• siRNA validation:

  • Transfect cells with PDE5A-specific siRNA

  • Confirm knockdown by qPCR

  • Verify reduction in antibody signal by Western blot/immunostaining

• Multiple antibody validation:

  • Use antibodies targeting different PDE5A epitopes

  • Compare staining patterns and signal intensity

  • Consistent results across antibodies support specificity

• Expected signal characteristics:

  • Western blot: ~100 kDa band in human and bovine tissues

  • IHC/IF: Primarily cytoplasmic localization

  • Subcellular fractionation: Enrichment in caveolin-rich lipid raft fractions

How do I troubleshoot weak or non-specific PDE5A antibody signals?

Common issues and solutions:

For Western blot:

• Weak signal:

  • Increase antibody concentration (try 1:500 instead of 1:1000)

  • Extend primary antibody incubation time to overnight at 4°C

  • Increase protein loading (50-75 μg)

  • Use more sensitive detection methods (e.g., chemiluminescent substrate with longer exposure)

• High background:

  • Use 5% BSA instead of milk for antibody dilution

  • Extend blocking time to 2 hours

  • Increase wash duration and number (5 × 10 minutes)

  • Reduce secondary antibody concentration

For IHC/IF:

• Weak or absent signal:

  • Optimize antigen retrieval (test both citrate and EDTA buffers)

  • Reduce fixation time in future specimens

  • Increase antibody concentration (1:100 instead of 1:200)

  • Try signal amplification systems (e.g., tyramide signal amplification)

• Non-specific staining:

  • Extend blocking time to 1-2 hours

  • Add 1-5% BSA to blocking solution

  • Pre-absorb secondary antibody with tissue powder

  • Use more stringent washing conditions

• Autofluorescence issues (for IF):

  • Include Sudan Black B treatment (0.1% in 70% ethanol for 20 minutes)

  • Use TrueBlack® Lipofuscin Autofluorescence Quencher

  • Switch to longer wavelength fluorophores (red/far-red)

What are the best practices for quantifying PDE5A expression in experimental samples?

For accurate quantification of PDE5A:

• Western blot densitometry:

  • Include loading controls (β-actin, GAPDH, or vinculin)

  • Use proper normalization: PDE5A signal ÷ loading control signal

  • Include a standard curve of recombinant PDE5A for absolute quantification

  • Analyze multiple exposure times to ensure signal is in linear range

• Immunohistochemistry quantification:

  • Use digital image analysis software (ImageJ, QuPath, etc.)

  • Score both staining intensity and percentage of positive cells

  • Calculate H-score: Σ(intensity × percentage) ranging from 0-300

  • Analyze multiple fields per sample (minimum 5-10)

  • Use automated algorithms to reduce observer bias

• qPCR for transcriptional analysis:

  • Design primers specific to PDE5A (avoid cross-reactivity with other PDEs)

  • Normalize to stable reference genes (GAPDH, β-actin, 18S rRNA)

  • Calculate fold changes using 2^(-ΔΔCt) method

  • Include melt curve analysis to verify amplicon specificity

• Enzymatic activity assays:

  • Measure cGMP-dependent PDE activity

  • Use selective PDE5A inhibitors to determine PDE5A-specific contribution

  • In endothelial cells, PDE5A contributes approximately 40% of total cGMP-dependent PDE activity