Phospho-PRKCQ (Ser695) Antibody

What is Phospho-PRKCQ (Ser695) Antibody and what does it specifically detect?

Phospho-PRKCQ (Ser695) Antibody, also known as Phospho-PKC theta (Ser695) Antibody, is a polyclonal antibody that specifically detects endogenous levels of PKC theta protein only when phosphorylated at serine 695. The antibody recognizes the peptide sequence around the phosphorylation site of serine 695 (N-F-S(p)-F-M) derived from Human PKC-theta . This specificity makes it valuable for studying the phosphorylation status of PKC theta in various signaling pathways, particularly in T-cell receptor (TCR) signaling.

What are the validated applications for Phospho-PRKCQ (Ser695) Antibody?

Phospho-PRKCQ (Ser695) Antibody has been validated for multiple research applications:

293 cells are suggested as a positive control for Western blot, while human lung carcinoma tissue is recommended as a positive control for IHC applications .

How should Phospho-PRKCQ (Ser695) Antibody be stored and handled for optimal stability?

For optimal stability and performance, Phospho-PRKCQ (Ser695) Antibody should be stored at -20°C for long-term storage (up to one year from the date of receipt). For frequent use and short-term storage, the antibody can be kept at 4°C for up to one month. It's crucial to avoid repeated freeze-thaw cycles as they can degrade the antibody and reduce its efficacy . The antibody is typically supplied in liquid form in PBS containing 50% glycerol, 0.5% BSA and 0.02% sodium azide at pH 7.4, which helps maintain stability during storage .

How can I optimize Western blot protocols when using Phospho-PRKCQ (Ser695) Antibody?

Optimizing Western blot protocols for Phospho-PRKCQ (Ser695) Antibody requires attention to several key factors:

• Sample preparation: Treat cells with appropriate stimuli (e.g., EGF 200ng/ml for 15 minutes in Jurkat cells) to induce phosphorylation at Ser695 .

• Lysate preparation: Use phosphatase inhibitors in your lysis buffer to preserve phosphorylation status.

• Loading controls: Include both phosphorylated and non-phosphorylated samples to demonstrate specificity.

• Blocking: Use 5% BSA rather than milk for blocking, as milk contains phosphoproteins that may interfere with detection.

• Antibody dilution: Start with a 1:1000 dilution and adjust based on signal strength and background .

• Validation controls: Consider using phosphopeptide blocking to confirm specificity. The lane blocked with phosphopeptide should show significantly reduced signal compared to the unblocked sample .

Recommended positive controls include 293 cells or Jurkat cells treated with EGF, which have demonstrated consistent results in previous studies .

What considerations are important when designing immunohistochemistry experiments with Phospho-PRKCQ (Ser695) Antibody?

When designing immunohistochemistry experiments with Phospho-PRKCQ (Ser695) Antibody, consider these methodological approaches:

• Tissue preparation: Use 4% paraformaldehyde fixation followed by paraffin embedding for optimal epitope preservation.

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is typically effective for phospho-epitopes.

• Blocking: Use species-appropriate serum (typically goat serum) with PBS containing 0.1% Triton X-100 for 1 hour at room temperature.

• Antibody dilution: Begin with 1:100 dilution in blocking buffer and optimize as needed .

• Incubation conditions: Overnight incubation at 4°C typically yields optimal results.

• Controls: Include phosphopeptide-blocked controls to confirm specificity. Human breast carcinoma and human lung carcinoma tissues have been successfully used as positive controls .

• Detection system: Use a sensitive detection system compatible with rabbit primary antibodies, such as HRP-conjugated secondary antibodies with DAB substrate.

• Counterstaining: Light hematoxylin counterstaining allows visualization of tissue architecture without obscuring specific staining.

How can Phospho-PRKCQ (Ser695) be used to study T-cell activation and signaling pathways?

Phospho-PRKCQ (Ser695) antibodies can be instrumental in studying T-cell activation and signaling through multiple methodological approaches:

• Phosphorylation kinetics analysis: Monitor the temporal dynamics of PRKCQ Ser695 phosphorylation following T-cell receptor (TCR) engagement to map activation pathways. This can be achieved through time-course Western blotting following stimulation with anti-CD3/CD28 antibodies.

• Signaling pathway mapping: Use the antibody to determine how Ser695 phosphorylation relates to other phosphorylation events (like Thr-538 and Ser-676) and downstream effector activation .

• Functional correlation studies: Correlate phosphorylation at Ser695 with functional outputs such as cytokine production (particularly IL-2), T-cell proliferation, and differentiation.

• Subcellular localization: Use immunofluorescence with Phospho-PRKCQ (Ser695) antibody to track the translocation of phosphorylated PRKCQ to the immunological synapse during T-cell activation .

• Inhibitor studies: Assess the impact of various kinase inhibitors on Ser695 phosphorylation to identify upstream regulators.

This approach can provide insights into how PRKCQ phosphorylation regulates multiple transcription factors such as NF-kappa-B, JUN, NFATC1, and NFATC2, which are essential for T-cell activation and function .

What methodologies can be employed to deliver anti-Phospho-PRKCQ antibodies into cells for functional studies?

Intracellular delivery of anti-Phospho-PRKCQ antibodies presents a significant challenge that can be addressed through several advanced methodological approaches:

• Protein transduction domain mimics (PTDMs): These synthetic polymers can efficiently complex with antibodies and facilitate their cellular entry. For example, P13D5 has been successfully used to deliver anti-pPKCθ antibodies into human peripheral mononuclear blood cells (hPBMCs) with approximately 60% transfection efficiency .

• Commercial transfection reagents: Products like AbDeliverIN can be used, though their efficiency may be lower than specialized PTDMs for certain cell types like hPBMCs .

• Electroporation: This technique can be optimized for hard-to-transfect cells, though it may affect cell viability.

• Microinjection: For single-cell studies where precision is critical.

The PTDM approach has demonstrated particular promise, with antibody detection possible for up to 72 hours post-delivery, enabling extended functional studies. When properly delivered, these antibodies can modulate downstream signaling events, including affecting CARMA1 phosphorylation, NOTCH1 cleavage, and nuclear localization of PKCθ .

How does phosphorylation at Ser695 interact with other post-translational modifications of PRKCQ in regulating its function?

PRKCQ undergoes multiple phosphorylation events that act in concert to regulate its function. The interrelationship between these modifications can be studied using the following methodological approaches:

• Sequential immunoprecipitation: First immunoprecipitate with one phospho-specific antibody, then probe the immunoprecipitate with antibodies against other phosphorylation sites to determine co-occurrence.

• Site-directed mutagenesis: Generate PRKCQ constructs with mutations at Ser695 and other phosphorylation sites (Thr-538, Ser-676) to assess functional interdependence.

• Phosphatase treatment assays: Selective dephosphorylation followed by functional assays can reveal the hierarchy of phosphorylation events.

• Phosphorylation kinetics: Compare the temporal sequence of phosphorylation at different sites following T-cell activation.

Research has shown that autophosphorylation at Thr-219 is required for targeting to the TCR and cellular function of PRKCQ upon antigen receptor ligation. Following TCR stimulation, phosphorylation occurs at both Tyr-90 and Ser-685 . While Ser695 phosphorylation is critical, Ser676 autophosphorylation also plays a significant role in PRKCQ activity . The precise interplay between these sites is still being elucidated, but evidence suggests a sequential phosphorylation model where certain modifications are prerequisites for others .

What are the common causes of non-specific binding when using Phospho-PRKCQ (Ser695) Antibody, and how can they be addressed?

Non-specific binding when using Phospho-PRKCQ (Ser695) Antibody can arise from several sources that require systematic troubleshooting:

• Antibody concentration: Excessive antibody concentration is a common cause of background. Perform a titration experiment (1:500, 1:1000, 1:2000) to determine optimal dilution for your specific application .

• Blocking inefficiency: Inadequate blocking can lead to high background. For phospho-specific antibodies, use 5% BSA in TBST rather than milk proteins, which contain phosphoproteins that may interfere with specificity.

• Cross-reactivity: Though the antibody is designed to be specific, it may recognize structurally similar phospho-epitopes. Validate specificity using:

  • Phosphopeptide competition assays with the immunizing peptide (N-F-S(p)-F-M)

  • Samples treated with lambda phosphatase as negative controls

  • PRKCQ-knockout or knockdown samples

• Fixation artifacts: In IHC applications, overfixation can create artifacts. Optimize fixation time and consider different antigen retrieval methods.

• Detection system sensitivity: If using a highly sensitive detection system, reduce antibody concentration accordingly or shorten substrate development time.

Each experiment should include both positive controls (293 cells, Jurkat cells treated with EGF, human lung carcinoma) and negative controls (phosphopeptide-blocked antibody, secondary antibody only) .

How can I validate the specificity of Phospho-PRKCQ (Ser695) Antibody in my experimental system?

Validating the specificity of Phospho-PRKCQ (Ser695) Antibody requires a multi-pronged approach:

• Phosphopeptide competition: Pre-incubate the antibody with excess phosphopeptide containing the Ser695 phosphorylation site (N-F-S(p)-F-M). A specific antibody will show significantly reduced or absent signal in Western blot, IHC, or IF applications when blocked with the phosphopeptide .

• Phosphatase treatment: Treat one sample with lambda phosphatase to remove phosphate groups. A phospho-specific antibody should show diminished or no signal in the dephosphorylated sample.

• Stimulation-dependent phosphorylation: Compare samples from resting cells versus those stimulated with agents known to induce PRKCQ phosphorylation (e.g., EGF treatment of Jurkat cells) .

• Genetic approaches: Use PRKCQ knockout/knockdown models or cells expressing phospho-deficient mutants (S695A) as negative controls.

• Multiple techniques: Confirm findings using at least two different techniques (e.g., Western blot and IHC) to rule out technique-specific artifacts.

• Testing related phosphorylation sites: Confirm the antibody doesn't cross-react with other phosphorylation sites (e.g., Ser676, Thr538) by using phosphopeptides for these sites in competition assays .

These validation steps should be performed for each new lot of antibody and in each experimental system to ensure reliable results.

How can Phospho-PRKCQ (Ser695) Antibody be used to investigate the role of PRKCQ in inflammatory and autoimmune disorders?

Investigating PRKCQ's role in inflammatory and autoimmune disorders using Phospho-PRKCQ (Ser695) Antibody can be approached through these methodological frameworks:

• Tissue expression profiling: Compare phosphorylation patterns in healthy versus diseased tissues using IHC. Particular focus should be given to tissues with high T-cell infiltration, such as inflamed intestinal mucosa in inflammatory bowel disease .

• Ex vivo cell analysis: Isolate peripheral blood mononuclear cells (PBMCs) or tissue-resident immune cells from patients with autoimmune conditions and healthy controls. Analyze basal and stimulation-induced phosphorylation patterns by flow cytometry or Western blot.

• Functional correlation studies: Correlate the degree of Ser695 phosphorylation with:

  • Production of pro-inflammatory cytokines (IL-17, IL-2)

  • T-helper cell differentiation (Th1, Th2, Th17)

  • Disease activity indices

• Intervention studies: Utilize the intracellular delivery of anti-Phospho-PRKCQ (Ser695) antibodies via protein transduction domain mimics (PTDMs) to modulate T-cell activation in disease models .

• Signaling pathway analysis: Investigate how PRKCQ phosphorylation at Ser695 influences downstream events critical in autoimmunity, such as CARD11 phosphorylation, NF-κB activation, and IL-17 production .

This approach is particularly relevant as PRKCQ plays an important role in the development of T-helper 2 (Th2) cells following immune and inflammatory responses, and is necessary for the activation of IL17-producing Th17 cells implicated in inflammatory autoimmune diseases .

What are the methodological considerations for using Phospho-PRKCQ (Ser695) Antibody in studying PRKCQ's role in different T-cell subsets?

Studying PRKCQ's differential roles across T-cell subsets requires specialized methodological approaches when using Phospho-PRKCQ (Ser695) Antibody:

• Cell isolation and purification: Use magnetic or fluorescence-activated cell sorting to isolate specific T-cell subsets (CD4+ naïve, Th1, Th2, Th17, Treg, CD8+ naïve, effector, memory) before analysis.

• Flow cytometry adaptation: For intracellular phospho-flow cytometry:

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with methanol or specialized permeabilization buffers

  • Co-stain with subset-defining markers (CD4, CD8, CD45RA, CCR7, etc.)

  • Use appropriate dilution (typically 1:50-1:100) of Phospho-PRKCQ (Ser695) Antibody

  • Include phosphopeptide-blocked controls

• Differentiation assays: Induce differentiation of naïve T-cells into various subsets in vitro, then analyze Ser695 phosphorylation kinetics during the differentiation process.

• Subset-specific functional correlations: Correlate Ser695 phosphorylation with:

  • Th1: IFN-γ production, T-BET expression

  • Th2: IL-4 production, GATA3 expression

  • Th17: IL-17 production, RORγt expression

  • Treg: Suppressive function, FOXP3 stability

• Compartmentalization studies: Compare membrane, cytoplasmic, and nuclear fractions for differential localization of phosphorylated PRKCQ across T-cell subsets .

This approach is particularly important as PRKCQ has been shown to have different roles across T-cell subsets, with particularly strong effects on Th2 and Th17 development, while potentially playing a more minor role in Th1 responses .

What emerging technologies might enhance the utility of Phospho-PRKCQ (Ser695) Antibody in single-cell analyses?

Several emerging technologies could significantly enhance the utility of Phospho-PRKCQ (Ser695) Antibody for single-cell analyses:

• Mass cytometry (CyTOF): Adapting Phospho-PRKCQ (Ser695) Antibody for metal-conjugation would allow simultaneous detection of numerous phosphorylation sites and cellular markers without fluorescence spectral overlap limitations. This could reveal heterogeneity in PRKCQ signaling across immune cell subpopulations.

• Imaging mass cytometry: This technology would enable spatial analysis of Phospho-PRKCQ (Ser695) within tissue microenvironments, particularly at the immunological synapse and in T-cell interactions within lymphoid tissues.

• Proximity ligation assays (PLA): Combining Phospho-PRKCQ (Ser695) Antibody with antibodies against interaction partners could visualize protein-protein interactions dependent on Ser695 phosphorylation at the single-molecule level.

• Single-cell phosphoproteomics: Integrating antibody-based enrichment of phosphorylated PRKCQ prior to single-cell mass spectrometry could provide comprehensive phosphorylation landscapes.

• Advanced intracellular delivery systems: Further development of protein transduction domain mimics (PTDMs) could enhance the delivery efficiency of functional anti-Phospho-PRKCQ antibodies into specific immune cell subsets for targeted modulation .

• CRISPR-based phospho-sensors: Engineering cellular reporters that link PRKCQ Ser695 phosphorylation status to fluorescent or luminescent outputs could enable real-time monitoring in living cells.

These approaches would move beyond population-level analyses to reveal cell-to-cell variability in PRKCQ signaling, potentially identifying previously unrecognized T-cell functional states relevant to immune regulation and disease.

How might comparative analysis of different PRKCQ phosphorylation sites advance our understanding of its regulation?

Comparative analysis of PRKCQ phosphorylation sites (including Ser695, Thr538, Ser676, Thr219, Tyr90, and Ser685) could significantly advance our understanding of PRKCQ regulation through these methodological approaches:

• Temporal sequence mapping: Design time-course experiments using antibodies against different phosphorylation sites to establish the chronological order of phosphorylation events following T-cell activation. This could reveal regulatory hierarchies and feedback mechanisms.

• Phosphorylation interdependence: Develop phospho-mimetic and phospho-deficient mutants for each site to determine how modification at one site affects others. For example, examining whether Thr538 phosphorylation is prerequisite for Ser695 phosphorylation.

• Kinase-substrate relationship mapping: Identify the specific kinases responsible for each phosphorylation site through kinase inhibitor screening, in vitro kinase assays, and mass spectrometry-based approaches.

• Structural biology integration: Use structural analysis to understand how phosphorylation at different sites induces conformational changes that affect protein-protein interactions and catalytic activity.

• Site-specific functional assays: Develop assays to distinguish the functional consequences of phosphorylation at different sites. For instance, comparing how Ser695 versus Ser676 phosphorylation differentially affects:

  • Substrate specificity

  • Cellular localization

  • Protein stability

  • Interaction with adaptor proteins