PDE8A Antibody, HRP conjugated

What is PDE8A and why is it an important research target?

PDE8A (phosphodiesterase 8A) is an enzyme that plays a crucial role in regulating intracellular levels of cAMP. It belongs to the cyclic nucleotide phosphodiesterase family and specifically to the PDE8 subfamily. PDE8A is involved in a wide range of physiological processes, including cell growth, hormonal regulation, and neurotransmission. Dysregulation of PDE8A has been implicated in various diseases, such as cancer, cardiovascular disorders, and neurodegenerative conditions .

PDE8A is particularly notable for:

• Its high affinity for cAMP

• Being IBMX-insensitive (unlike most other PDEs)

• Containing a distinctive PAS (Per-Arnt-Sim) domain that mediates protein-protein interactions

• Having a molecular weight of approximately 93 kDa

What are the primary applications for HRP-conjugated PDE8A antibodies?

HRP-conjugated PDE8A antibodies are versatile tools used in several key research applications:

The HRP conjugation allows for direct detection without the need for secondary antibodies, simplifying experimental workflows and potentially reducing background signals. The enzyme reacts with substrates like TMB to produce colorimetric or chemiluminescent signals proportional to antibody binding .

What sample types can be successfully analyzed with PDE8A antibodies?

PDE8A antibodies have been validated for use with various biological samples:

• Human serum and plasma (including citrated/EDTA plasma)

• Cell culture supernatants

• Mouse tissues (particularly testis, spleen, eye, and liver)

• Rat tissues (particularly testis)

• Human tissues (including colon cancer tissue)

• Cell lines (such as HEK293, SH-SY5Y)

For optimal results with different sample types, appropriate sample preparation, including proper lysis buffers and protein extraction methods, should be employed depending on the application.

How should HRP-conjugated PDE8A antibodies be stored and handled for optimal activity?

For maintaining optimal activity of HRP-conjugated PDE8A antibodies:

• Store at -20°C in aliquots to prevent multiple freeze-thaw cycles

• Most formulations remain stable for one year after shipment when properly stored

• Use storage buffers containing stabilizers (typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3)

• Some small volume formulations (e.g., 20μl) may contain 0.1% BSA as an additional stabilizer

• Avoid repeated freeze-thaw cycles which can lead to loss of activity

• When working with the antibody, keep it on ice and return to -20°C promptly after use

What are common troubleshooting strategies for weak or non-specific signals with PDE8A antibodies?

When facing weak or non-specific signals in PDE8A detection:

• For weak signals:

  • Increase antibody concentration (within recommended ranges)

  • Extend incubation times

  • Ensure sample contains adequate PDE8A (mouse testis, spleen, and SH-SY5Y cells show good expression)

  • Check substrate freshness and development time

  • Use enhanced chemiluminescent substrates for Western blots

• For non-specific signals:

  • Optimize blocking conditions (5% non-fat milk or BSA)

  • Increase washing steps duration and frequency

  • Reduce primary antibody concentration

  • Include appropriate controls (knockout or knockdown samples)

  • Consider pre-absorbing antibody with non-specific proteins

• For immunohistochemistry applications:

  • Test different antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Optimize antibody dilution (1:20-1:200 range is recommended)

  • Use positive control tissues (human colon cancer tissue has shown good results)

How can PDE8A antibodies be used to study protein-protein interactions in signaling pathways?

PDE8A has been shown to interact with several important signaling proteins, including Raf-1 kinase and IκB proteins. HRP-conjugated antibodies can be valuable tools for studying these interactions:

• Immunoprecipitation followed by Western blotting:

  • Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

  • Immunoprecipitate PDE8A and probe for interacting partners, or vice versa

  • HRP-conjugated antibodies simplify detection in these complex experiments

• PAS domain-mediated interactions:

  • The PAS domain of PDE8A is crucial for its interaction with IκBβ

  • Experiments comparing wild-type PDE8A with PAS-deleted PDE8A (ΔPAS) demonstrate the domain specificity of these interactions

  • Custom antibody arrays can be used to screen for PAS domain-dependent interactions

• Studying the PDE8A-Raf-1 complex:

  • The interaction between PDE8A and Raf-1 has been shown to be extremely high affinity (Kd <61 pM)

  • This complex plays a role in regulating MAPK signaling pathways

  • Disruption of this complex using specific peptides affects EGF-induced morphological changes and ERK signaling

What methodological considerations are important when using PDE8A antibodies to study cAMP compartmentalization?

PDE8A plays a critical role in modulating specific compartments of cAMP and PKA activity. When studying these compartmentalized signaling events:

• Spatial resolution techniques:

  • Combine PDE8A antibodies with FRET-based cAMP sensors for live-cell imaging

  • Use subcellular fractionation followed by immunoblotting to track PDE8A localization

  • Consider proximity ligation assays to visualize PDE8A interactions with other proteins in situ

• Functional studies:

  • Compare effects of global cAMP elevation (e.g., with forskolin) versus targeted PDE8A inhibition

  • Use PDE8A-specific inhibitors (like dipyridimole) in conjunction with antibody-based detection

  • The PDE8A-Raf-1 complex generates a localized compartment where PKA-mediated inhibitory phosphorylation on Ser259 of Raf-1 is reduced through local cAMP hydrolysis

• Dynamic regulation:

  • PDE8A interacts with 14-3-3ζ, and this interaction is enhanced upon PKA activation

  • This interaction provides a potential mechanism for regulating PDE8A's effects on localized cAMP pools

  • Time-course experiments combining pharmacological interventions with antibody detection can reveal dynamic regulation

What are the considerations when using PDE8A antibodies in multiplex and advanced detection systems?

For multiplex and advanced detection approaches:

• Antibody compatibility:

  • Ensure that epitopes for different target proteins don't overlap when using multiple antibodies

  • Consider using different conjugates (HRP, fluorophores) for simultaneous detection

  • Test for cross-reactivity between antibodies in your system

• Detection sensitivity:

  • HRP-conjugated antibodies offer high sensitivity with ELISA assays (detection limit ~0.105 ng/ml)

  • Amplification systems (like tyramide signal amplification) can further enhance sensitivity

  • Advanced Western blot detection requires careful optimization of antibody dilution (1:500-1:2400 recommended for most systems)

• Cross-linking approaches:

  • Formaldehyde cross-linking (1%) has been shown to enhance detection of PDE8A interactions with proteins like 14-3-3

  • This approach can stabilize transient or weak interactions that might be missed in standard co-immunoprecipitation experiments

  • HRP-conjugated antibodies work well in these systems with proper optimization

How does the 14-3-3 interaction with PDE8A affect experimental approaches using PDE8A antibodies?

Recent research has revealed that PDE8A interacts with 14-3-3 proteins, with important implications for experimental approaches:

• Experimental detection:

  • Cross-linking with 1% formaldehyde substantially enhances detection of the PDE8A/14-3-3 interaction

  • This suggests that standard co-immunoprecipitation protocols may underestimate this interaction

  • Researchers should consider including cross-linking steps when studying PDE8A complexes

• Functional significance:

  • The 14-3-3 interaction with PDE8A is enhanced upon PKA activation

  • This provides a potential feedback mechanism whereby PKA regulates its own inhibition via PDE8A

  • Experiments studying PDE8A function should consider PKA activity status

• Conservation across species:

  • The interaction between human PDE8A and 14-3-3 proteins appears conserved, as demonstrated using yeast BMH1 and BMH2 proteins (yeast 14-3-3 homologs)

  • This conservation suggests fundamental importance of this regulatory mechanism

What methodological approaches are recommended when studying the PDE8A-Raf-1 complex using antibodies?

The PDE8A-Raf-1 complex is an important target for research with therapeutic implications:

• Complex isolation:

  • Immunoprecipitates of Raf-1 contain PDE8A and associated PDE activity

  • This activity is inhibited by dipyridimole, a PDE8 inhibitor

  • When detecting complexes, consider antibody orientation (anti-Raf-1 for IP followed by anti-PDE8A for detection, or vice versa)

• Experimental disruption:

  • Cell-permeable peptides based on the Raf-1-docking sequence from PDE8A (residues R454-T465) can disrupt the complex

  • Using such peptides alongside antibody detection can reveal functional consequences

  • Stearylated peptides with C-terminal stearate groups are particularly effective for cell permeability

• Functional readouts:

  • Disruption of the PDE8A-Raf-1 complex affects EGF-induced morphological changes and ERK signaling

  • Impedance measurements and phospho-ERK detection are valuable functional readouts

  • Catalytically inactive PDE8A constructs can be used as dominant-negative controls

What considerations are important when using PDE8A antibodies for detecting specific isoforms or variants?

PDE8A exists in multiple isoforms, which requires careful consideration in experimental design:

• Epitope selection:

  • Antibodies targeting internal regions of PDE8A may detect multiple isoforms

  • The observed molecular weight can vary (93 kDa and 88 kDa have been reported)

  • Researchers should validate which isoforms are detected by their specific antibody

• Domain-specific detection:

  • Antibodies can be designed to target specific domains, such as the PAS domain

  • Comparing results with domain-deleted constructs (e.g., ΔPAS-PDE8A) can provide domain-specific information

  • Recombinant fusion proteins containing specific sequences (e.g., amino acids 1-120 of human PDE8A) are often used as immunogens

• Species considerations:

  • PDE8A antibodies show varying reactivity across species

  • Most commercial antibodies are validated for human and mouse samples

  • Cross-reactivity testing is recommended when working with other species