Phospho-PRKD1/PRKD2/PRKD3 (Ser738/742) Antibody

What is the Phospho-PRKD1/PRKD2/PRKD3 (Ser738/742) Antibody and what does it detect?

The Phospho-PRKD1/PRKD2/PRKD3 (Ser738/742) Antibody is a polyclonal antibody typically raised in rabbits that specifically recognizes the phosphorylated serine residues at positions 738 and 742 (or their equivalent positions) in protein kinase D isoforms. This antibody detects the activation loop phosphorylation that is crucial for PKD activity regulation. The antibody binds to the conserved phosphorylation motif present in all three mammalian PKD isoforms: PKD1 (phosphorylated on Ser744/Ser748), PKD2 (phosphorylated on Ser707/Ser711), and PKD3 (phosphorylated on Ser730/Ser734) . The specificity of these antibodies is typically validated through various techniques including analysis in PKD isoform knockout models and alkaline phosphatase treatment.

What are the validated applications for this antibody?

The Phospho-PRKD1/PRKD2/PRKD3 (Ser738/742) Antibody has been validated for multiple research applications:

When using this antibody for Western blot applications, researchers should optimize the dilution based on their specific experimental conditions and sample types. The antibody typically detects bands at approximately 100-110 kDa, corresponding to the molecular weights of the PKD isoforms .

What species reactivity has been confirmed for this antibody?

The species reactivity profile has been established through experimental validation:

How can I validate the specificity of this antibody in my experimental system?

Validating antibody specificity is crucial for reliable research outcomes. For the Phospho-PRKD1/PRKD2/PRKD3 (Ser738/742) Antibody, consider these methodological approaches:

• Alkaline Phosphatase (AP) Treatment: Treat half of your sample with alkaline phosphatase to remove phosphate groups. Compare treated vs. untreated samples via Western blot - signal should disappear in the AP-treated sample .

• Peptide Competition Assay: Pre-incubate the antibody with the specific phosphopeptide used as the immunogen. This should block the antibody's ability to bind to the phosphorylated protein in your samples .

• Knockout/Knockdown Controls: Utilize cells with genetic deletion or knockdown of PRKD1/2/3. Compare with wild-type cells to confirm specificity .

• Stimulation/Inhibition Paradigms: Treat cells with known activators (e.g., phorbol esters like PMA) and/or inhibitors (e.g., Gö-6976, kb-NB142-70) of PKD signaling to demonstrate dynamic changes in phosphorylation .

The data in search result illustrates how researchers used PKD1-/- and PKD3-/- DT40 B-lymphocytes to validate the specificity of PKD activation loop phospho-specific antibodies, demonstrating that these antibodies can detect both PKD1 and PKD3 when phosphorylated.

What are the recommended storage and handling conditions for this antibody?

To maintain antibody integrity and performance:

• Long-term Storage: Store at -20°C, typically in aliquots to avoid repeated freeze-thaw cycles. Most preparations remain stable for at least one year under these conditions .

• Short-term Storage: For frequent use within a month, store at 4°C .

• Buffer Composition: The antibody is typically supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide . This formulation helps maintain stability during storage.

• Avoid Freeze-Thaw Cycles: Repeated freezing and thawing can degrade antibodies. Creating single-use aliquots upon receipt is recommended .

• Working Solution Preparation: Dilute only the amount needed for immediate experiments in appropriate buffer according to the application.

How does the antibody perform in distinguishing between different PRKD isoforms?

The Phospho-PRKD1/PRKD2/PRKD3 (Ser738/742) Antibody recognizes a conserved phosphorylation motif present in all three PKD isoforms. This cross-reactivity presents both advantages and challenges:

• Cross-reactivity Analysis: The antibody detects PKD1 (phosphorylated on Ser744/Ser748), PKD2 (phosphorylated on Ser707/Ser711), and PKD3 (phosphorylated on Ser730/Ser734) due to the high conservation of the activation loop segment across all three mammalian PKD isoforms .

• Quantitative Considerations: The research by Matthews et al. described in search result demonstrates that in lymphoid cells with different expression levels of PKD isoforms, the detection of specific isoforms depends on their relative abundance. In their study, PKD2 was the predominant isoform in murine lymphocytes, while PKD3 made minimal quantitative contribution despite being present.

• Immunoprecipitation Approach: To distinguish between isoforms, researchers can perform immunoprecipitation with isoform-specific antibodies followed by detection with the phospho-specific antibody. This approach was used successfully to detect low-abundance phosphorylated PKD3 in PKD2 mutant thymocytes .

• Knockout Model Analysis: The most definitive approach to determine isoform-specific signals is to use cells or tissues from knockout models lacking specific PKD isoforms, as demonstrated in the DT40 B-lymphocyte model system .

How does the phosphorylation at Ser738/742 relate to other phosphorylation sites in PRKD proteins?

PKD proteins undergo phosphorylation at multiple sites that regulate their activity and function:

• Activation Loop (Ser738/742): Phosphorylation at these sites by upstream PKCs is a critical step in PKD activation and is detected by the Phospho-PRKD1/PRKD2/PRKD3 (Ser738/742) Antibody .

• Autophosphorylation Sites:

  • Ser916 in PKD1 is an autophosphorylation site commonly used as a readout of PKD1 activity. The phosphorylation of this site can be detected using specific Phospho-PRKD1 (Ser916) antibodies .

  • Ser876 in PKD2 is an equivalent autophosphorylation site that is also used as an indicator of PKD2 activity .

• Other Regulatory Sites:

  • Ser205 in PKD1 is another phosphorylation site that may play a role in regulating PKD1 function .

  • Tyr438 and Tyr717 in PKD2 are phosphorylated by ABL1 in response to oxidative stress, leading to PKD2 activation without increasing its catalytic activity .

  • Ser244 in PKD2 is phosphorylated by CSNK1D and CSNK1E in response to gastrin receptor activation, leading to nuclear translocation .

The relationship between these phosphorylation events follows a sequential pattern: typically, activation loop phosphorylation (Ser738/742) precedes and enables autophosphorylation at sites like Ser916 or Ser876, which serves as an indicator of kinase activation and activity.

What methodological approaches can be used to study PKD signaling dynamics?

Researchers investigating PKD signaling dynamics can employ several sophisticated methodological approaches:

• Reverse Phase Protein Array (RPPA):

  • RPPA is particularly valuable for quantitative proteomic analysis from limited sample material such as patient tissues .

  • This technique allows for systematic phospho-protein profiling within cell signaling networks, especially useful for studying post-translational modifications .

  • A key optimization involves using lysis buffers compatible with alkaline phosphatase (AP) treatment to validate phospho-antibody specificity .

• Pharmacological Manipulation:

  • Stimulation with phorbol esters (e.g., PMA) activates PKC, leading to subsequent PKD activation loop phosphorylation .

  • Inhibition with PKD-specific inhibitors (kb-NB142-70) or less-specific PKC/PKD inhibitors (Gö-6976) allows for manipulation of the pathway to assess downstream effects .

• Phosphorylation Network Analysis:

  • Antibody microarrays measuring phospho-protein levels can identify broad phosphorylation changes induced by PKD activity .

  • In a study by Yang et al., 81 protein phosphorylations were identified that increased when PKD1 was active and decreased when inhibited, revealing connections to tumor progression regulators, MAP kinase and NF-κB components .

• Genetic Models:

  • Knock-in of phosphorylation site mutants (e.g., PKD2 SSAA/SSAA where activation loop serines are replaced with alanines) provides insights into phosphorylation-dependent functions .

  • Expression of kinase-dead mutants (e.g., K612W) can act as dominant negatives to assess the requirement for kinase activity .

How is PKD phosphorylation altered in cancer and other pathological conditions?

PKD phosphorylation status has significant implications in various disease contexts:

• Cancer Progression:

  • Antibody-based protein profiling revealed that PKD1 induced broad phosphorylation changes in epithelial dissemination models, including an inactivating phosphorylation of β-catenin .

  • PKD signaling has been implicated in regulating cell proliferation via MAPK1/3 (ERK1/2) signaling pathways that are frequently dysregulated in cancer .

  • PKD2 activation in response to oxidative stress leads to NF-kappa-B activation, which plays a crucial role in inflammation and cancer progression .

• Immune System Function:

  • Studies in PKD2 mutant lymphocytes have shown that PKD2 is required for proper T cell function, including cytokine production after TCR engagement .

  • The phosphorylation status of PKD proteins affects their ability to regulate HDAC7 nuclear export, which influences T cell development and function .

• Methodological Approaches for Clinical Samples:

  • The RPPA-based phospho-antibody characterization approach has been successfully applied to both fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) clinical specimens .

  • This methodology shows reproducibility and significant correlation with pathological markers in melanoma and lung cancer tissues, generating data that match clinical features .

What experimental controls should be included when studying PKD phosphorylation in different cellular contexts?

When investigating PKD phosphorylation across different cellular contexts, robust experimental controls are essential:

• Positive Controls:

  • Treatment with PKC activators such as phorbol esters (PMA) to induce PKD phosphorylation .

  • Insulin treatment in NIH/3T3 cells has been validated as a positive control for PKD phosphorylation .

• Negative Controls:

  • Alkaline phosphatase (AP) treatment of samples to remove phosphate groups and confirm phospho-specific antibody binding .

  • Peptide competition assays where the immunizing peptide blocks antibody binding .

• Genetic Controls:

  • Cell lines with known PKD isoform expression profiles (e.g., DT40 B-lymphocytes express PKD1 and PKD3 but not PKD2) .

  • Knockout or knockdown models lacking specific PKD isoforms to verify antibody specificity .

  • Cells expressing mutant PKD proteins where phosphorylation sites are mutated to non-phosphorylatable residues (e.g., serine to alanine mutations) .

• Inhibitor Controls:

  • Treatment with PKD inhibitors like kb-NB142-70 or PKC/PKD inhibitors like Gö-6976 to verify phosphorylation-dependent signals .

  • Kinase-dead mutants expressed as dominant negatives can serve as functional inhibitor controls .

• Sample Type Considerations:

  • When comparing results across different sample types (cell lines vs. primary tissues, FF vs. FFPE), appropriate validation is necessary as fixation and processing can affect phosphoepitope detection .

What are common issues encountered when using phospho-specific antibodies and how can they be addressed?

Researchers frequently encounter specific challenges when working with phospho-specific antibodies like the Phospho-PRKD1/PRKD2/PRKD3 (Ser738/742) Antibody:

• High Background Signals:

  • Cause: Insufficient blocking, non-specific binding, or suboptimal antibody dilution.

  • Solution: Optimize blocking conditions (5% BSA often works better than milk for phospho-epitopes), increase washing steps, and test a range of antibody dilutions (1:500-1:2000 for WB) .

• Weak or No Signal:

  • Cause: Low abundance of phosphorylated protein, epitope masking, or phosphatase activity during sample preparation.

  • Solution: Include phosphatase inhibitors in lysis buffers, increase protein loading, and consider enrichment strategies like immunoprecipitation before detection .

• Multiple Bands:

  • Cause: Cross-reactivity with other phospho-proteins, degradation products, or detection of multiple PKD isoforms.

  • Solution: Use isoform-specific immunoprecipitation followed by phospho-detection, include peptide competition controls, and optimize sample preparation to minimize proteolysis .

• Inconsistent Results Across Experiments:

  • Cause: Variations in cell stimulation protocols, sample handling, or phosphatase activity.

  • Solution: Standardize stimulation protocols, minimize time between cell harvesting and lysis, maintain samples at cold temperatures during processing, and use freshly prepared lysis buffers with phosphatase inhibitors .

• Poor Reproducibility in FFPE Tissues:

  • Cause: Fixation-induced epitope masking or degradation of phospho-epitopes.

  • Solution: Optimize antigen retrieval methods specifically for phospho-epitopes, consider validation approaches like those described in the RPPA-based phospho-antibody characterization workflow .

How can researchers optimize experimental design to study temporal dynamics of PKD phosphorylation?

To effectively capture the temporal dynamics of PKD phosphorylation:

• Time Course Experiments:

  • Design comprehensive time course experiments with appropriate intervals (e.g., 0, 5, 15, 30, 60 minutes, 2, 6, 24 hours) following stimulation.

  • Include both early time points to capture rapid phosphorylation events and later time points to assess sustained responses.

• Stimulation Protocol Optimization:

  • Different stimuli induce distinct phosphorylation kinetics - phorbol esters typically cause sustained PKD phosphorylation, while physiological stimuli like growth factors may induce more transient responses .

  • Consider dose-response relationships when designing stimulation protocols, as concentration can affect both magnitude and duration of phosphorylation.

• Dual Phosphorylation Analysis:

  • Monitor both activation loop phosphorylation (Ser738/742) and autophosphorylation sites (e.g., Ser916 for PKD1, Ser876 for PKD2) simultaneously to distinguish between PKD activation and activity .

  • The temporal relationship between these phosphorylation events can provide insights into PKD regulation mechanisms.

• Subcellular Fractionation:

  • PKD proteins translocate between cellular compartments upon activation, so fractionation approaches can reveal spatial aspects of phosphorylation dynamics .

  • Combining temporal sampling with subcellular fractionation provides a comprehensive view of PKD signaling dynamics.

• Phosphatase Inhibition Controls:

  • Include parallel samples with and without phosphatase inhibitors to assess the contribution of dephosphorylation to the observed dynamics.

  • This approach can distinguish between cessation of phosphorylation versus active dephosphorylation mechanisms.