PRKD1 Antibody, FITC conjugated

What is PRKD1 and what cellular functions does it regulate?

PRKD1 (Protein Kinase D1) is a serine/threonine kinase that converts transient diacylglycerol (DAG) signals into prolonged physiological effects downstream of PKC. This 102 kDa protein (calculated molecular weight) plays crucial roles in multiple cellular processes including oxidative stress response through activation of NF-kappa-B, cell adhesion, cell migration, vesicle transport, and cell survival pathways . PRKD1 is expressed ubiquitously and is involved in diverse signaling cascades that regulate critical cellular functions . At the subcellular level, PRKD1 is primarily localized to the cytoplasm and cell membrane, where it can respond to various signaling inputs and phosphorylate downstream targets .

Validation studies have confirmed successful detection of PRKD1 in specific cell types:

Successful visualization requires proper sample preparation, with documented protocols using both 4% PFA fixation and -20°C ethanol fixation methods depending on cell type .

How should PRKD1 antibodies be stored and handled for maximum stability?

For optimal stability and performance, PRKD1 antibodies should be stored at -20°C . The antibodies are typically supplied in storage buffers containing components that enhance stability:

To maintain antibody performance, it is recommended to prepare multiple small aliquots upon receipt to minimize repeated freeze-thaw cycles, which can degrade antibody quality . The 20μL size of 83174-5-RR contains 0.1% BSA for added stability .

How can I optimize PRKD1 intracellular staining for flow cytometry analysis?

Intracellular staining of PRKD1 for flow cytometry requires careful optimization of fixation, permeabilization, and antibody concentration. Based on validated protocols:

• Harvest cells (approximately 1×10^6 cells per condition)

• Wash cells with ice-cold PBS containing 1% FBS

• Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature

• Wash twice with PBS containing 1% FBS

• Permeabilize cells using Flow Cytometry Perm Buffer (such as product PF00011-C)

• Incubate with primary PRKD1 antibody (83174-5-RR) at 0.25 μg per 10^6 cells in 100 μl suspension

• Incubate at room temperature for 30-60 minutes or at 4°C for 1-2 hours

• Wash twice with permeabilization buffer

• For indirect detection, incubate with fluorophore-conjugated secondary antibody (such as APC-Conjugated AffiniPure Goat Anti-Rabbit IgG)

• Analyze on flow cytometer using appropriate channels for FITC or secondary fluorophore

Always include appropriate isotype controls (0.25 μg of matched isotype antibody) to establish proper gating and distinguish specific from non-specific staining .

What methodological approaches can resolve detection issues in PRKD1 immunofluorescence experiments?

When troubleshooting PRKD1 detection by immunofluorescence, consider these methodological refinements:

• Fixation method optimization: Different cell types respond optimally to specific fixation methods. For HeLa cells, 4% PFA fixation has been validated, while LNCaP cells show better results with -20°C ethanol fixation .

• Antibody titration: Test a range of dilutions (1:50-1:500) to determine optimal signal-to-noise ratio for your specific samples .

• Signal amplification strategies: For low abundance targets, use a two-step detection method with primary antibody followed by fluorophore-conjugated secondary antibody (such as CoraLite®488-Conjugated AffiniPure Goat Anti-Rabbit IgG) .

• Permeabilization optimization: Adjust permeabilization conditions based on subcellular localization. Since PRKD1 localizes to both cytoplasm and cell membrane, ensure your permeabilization protocol maintains cellular morphology while allowing antibody access .

• Blocking optimization: Increase blocking time and concentration (typically 5-10% normal serum) to reduce background if non-specific binding occurs.

• Counterstaining: Include nuclear counterstains like DAPI to help visualize cellular context and confirm subcellular localization patterns.

How does PRKD1 function in cell cycle regulation and what methodologies can investigate this mechanism?

PRKD1 functions as a tumor and metastasis suppressor by influencing cell cycle progression, specifically inducing G1-phase cell-cycle arrest independent of checkpoint kinases (CHEKs) . Research methodologies to investigate this mechanism include:

• Cell cycle analysis: Flow cytometry with propidium iodide staining of cells with PRKD1 overexpression, knockdown, or pharmacological inhibition to quantify cell cycle distribution.

• In vitro phosphorylation assays: PRKD1 directly phosphorylates all CDC25 isoforms, which are critical cell cycle regulators. In vitro kinase assays with recombinant PRKD1 and CDC25 can confirm direct phosphorylation .

• Western blot analysis: Detection of phosphorylated CDC25 isoforms using phospho-specific antibodies in the presence or absence of PRKD1 activity.

• Real-time cell cycle monitoring: Using fluorescent cell cycle indicators in live cells with manipulated PRKD1 expression.

• Genetic rescue experiments: Introducing phosphorylation-resistant CDC25 mutants in PRKD1-expressing cells to determine if this overcomes the G1 arrest phenotype .

This represents an important area of investigation as it reveals a molecular mechanism distinct from CHEK kinases by which PRKD1 influences cell cycle progression and potentially suppresses tumorigenesis.

What protein-protein interactions does PRKD1 participate in and how can these be studied using PRKD1 antibodies?

PRKD1 engages in several critical protein-protein interactions that mediate its signaling functions. These interactions can be studied using PRKD1 antibodies through the following methodologies:

• Co-immunoprecipitation (Co-IP): PRKD1 antibodies can be used to pull down PRKD1 and its interacting partners. Studies have successfully employed this approach to investigate interactions in signaling pathways such as Toll-Like Receptor 9 (TLR9) signaling, where FLAG-tagged PKD1 was used to detect interactions with TLR9, MyD88, IRAK1, IRAK4, TRAF6, and TRAF3 .

• Immunofluorescence co-localization: FITC-conjugated PRKD1 antibodies enable direct visualization of PRKD1 co-localization with potential interacting proteins using confocal microscopy and quantitative co-localization analysis.

• Proximity ligation assay (PLA): This technique allows detection of protein interactions in situ with greater sensitivity than conventional co-localization methods.

• FRET (Förster Resonance Energy Transfer): Using FITC-conjugated PRKD1 antibodies paired with secondary antibodies against potential interaction partners labeled with appropriate acceptor fluorophores.

• Transcription complex analysis: Studies have shown that PRKD1 influences β-catenin/TCF-4 transcription complex formation, which can be analyzed by immunoprecipitating the complex and probing for PRKD1, β-catenin, and TCF-4 .

The PRKD1 antibody can be particularly useful for investigating how PRKD1 interacts with components of the Wnt signaling pathway, as research has demonstrated that PRKD1 attenuates tumorigenesis in colon cancer by modulating this pathway .

How can PRKD1 antibodies be employed to study its role in oncogenic transformation and tumor suppression?

PRKD1 has documented roles in both oncogenic transformation (when mutated) and tumor suppression (in its wild-type form), making it an important target for cancer research. PRKD1 antibodies can be employed in the following methodological approaches:

• Mutation-specific detection: Developing and utilizing antibodies that specifically recognize the p.Glu710Asp hotspot mutation found in 72.9% of polymorphous low-grade adenocarcinomas (PLGAs) .

• Tissue microarray analysis: Using FITC-conjugated PRKD1 antibodies to assess expression levels across multiple tumor types and correlate with clinical outcomes and molecular profiles.

• Functional domain mapping: Employing domain-specific PRKD1 antibodies to understand how different regions contribute to tumor suppression or oncogenic functions when mutated.

• In vivo tumor models: Using PRKD1 antibodies for immunohistochemical analysis of tumor tissues from animal models with manipulated PRKD1 expression or activity.

• Cell signaling pathway analysis: Investigating how PRKD1 influences oncogenic pathways by examining downstream targets after manipulation of PRKD1 in cancer cell lines.

Studies have shown that PRKD1 expression is negatively regulated by androgen receptor (AR) in prostate cancer, and PRKD1 functions as a tumor suppressor in colon cancer by attenuating Wnt/β-catenin signaling . These findings suggest that PRKD1 antibodies can be valuable tools for investigating cancer-specific signaling mechanisms.

What controls should be included when using FITC-conjugated PRKD1 antibodies?

When utilizing FITC-conjugated PRKD1 antibodies, proper controls are essential for experimental validity:

• Isotype control: Include a FITC-conjugated isotype-matched IgG (rabbit IgG for the antibodies discussed) at the same concentration as the PRKD1 antibody to assess non-specific binding and establish proper gating thresholds .

• Negative control samples: Include cells known not to express PRKD1 or samples where PRKD1 has been knocked down using siRNA/shRNA.

• Positive control samples: Include cells with confirmed PRKD1 expression such as HeLa or LNCaP cells, which have been validated in previous studies .

• Autofluorescence control: Include unstained samples to account for natural cellular fluorescence in the FITC channel.

• Absorption/quenching controls: When using multiple fluorophores, include single-stained controls to assess spectral overlap and establish appropriate compensation settings for flow cytometry or confocal microscopy.

• Secondary antibody control: If using a two-step detection method, include samples treated with secondary antibody only to assess non-specific binding of the secondary reagent.

These controls help establish specificity of staining and ensure accurate interpretation of results, particularly important when analyzing subtle differences in PRKD1 expression or localization across experimental conditions.

How can cross-reactivity with other PRKD family members be assessed and mitigated?

The PRKD family includes three closely related members (PRKD1, PRKD2, and PRKD3) with high sequence homology. To assess and mitigate cross-reactivity:

• Sequence alignment analysis: Compare the immunogen sequence used to generate the PRKD1 antibody against all PRKD family members to identify potential cross-reactive epitopes.

• Validation in knockout/knockdown systems: Test the antibody in cells where PRKD1 has been specifically knocked out or knocked down while PRKD2 and PRKD3 are still expressed.

• Recombinant protein array testing: Evaluate antibody binding to purified recombinant PRKD1, PRKD2, and PRKD3 proteins to quantify relative affinity.

• Co-immunoprecipitation specificity: Perform immunoprecipitation with the PRKD1 antibody followed by Western blot analysis using specific antibodies against each PRKD family member.

• Epitope mapping: Identify the specific epitope recognized by the antibody and assess its conservation across PRKD family members.

The recombinant PRKD1 antibody (83174-5-RR) is generated against a specific peptide sequence, potentially offering higher specificity than polyclonal alternatives . When studying PRKD1 in experimental systems where multiple PRKD family members are expressed, it is advisable to use recombinant monoclonal antibodies that have been validated for specificity.

What is the optimal protocol for dual-color immunofluorescence using FITC-conjugated PRKD1 antibody?

For dual-color immunofluorescence incorporating FITC-conjugated PRKD1 antibody with another protein of interest:

• Sample preparation:

  • Grow cells on coverslips or prepare tissue sections (4-5 μm thick)

  • Fix using validated method (4% PFA for HeLa cells or -20°C ethanol for LNCaP cells)

  • Permeabilize with 0.1-0.5% Triton X-100 in PBS for 10 minutes

• Blocking:

  • Block with 5-10% normal serum (matching species of secondary antibody for the non-FITC primary) in PBS with 0.1% Triton X-100 for 1 hour

• Primary antibody incubation:

  • Prepare antibody dilution mixture containing:

    • FITC-conjugated PRKD1 antibody (bs-21780R-FITC at 1:50-1:200)

    • Non-conjugated primary antibody for second target protein

  • Incubate overnight at 4°C in a humidified chamber

• Secondary antibody incubation:

  • Wash 3x with PBS

  • Incubate with secondary antibody for the non-FITC primary (choose a fluorophore with minimal spectral overlap with FITC, such as Cy3, Cy5, or Alexa 647)

  • Incubate for 1-2 hours at room temperature in the dark

• Nuclear counterstaining and mounting:

  • Wash 3x with PBS

  • Counterstain with DAPI (1 μg/ml) for 5 minutes

  • Mount with anti-fade mounting medium

• Imaging:

  • Use appropriate filter sets for FITC (excitation ~495 nm, emission ~520 nm)

  • Capture separate channels sequentially to minimize bleed-through

  • Include single-stained controls for spectral unmixing if needed

When choosing a second target protein, consider markers relevant to PRKD1 function, such as β-catenin for studies of Wnt signaling modulation or cell cycle markers for studies of G1 arrest mechanisms .

How can phospho-specific events in PRKD1 signaling be detected using combination of antibodies?

Detecting phosphorylation-dependent PRKD1 signaling events requires specialized methodological approaches:

• Sequential immunostaining protocol:

  • First detect total PRKD1 using FITC-conjugated antibody

  • Then detect phosphorylated substrates using phospho-specific antibodies

  • This approach allows correlation between PRKD1 localization and substrate phosphorylation

• Validated phospho-targets to monitor:

  • CDC25 isoforms: PRKD1 directly phosphorylates these cell cycle regulators

  • HDAC7: Phosphorylation by PRKD1 mediates its nuclear export

  • NF-κB pathway components: PRKD1 activates this pathway in response to oxidative stress

• Stimulus-response experimental design:

  • Baseline: Unstimulated cells stained for PRKD1-FITC and phospho-targets

  • Stimulation: Treat with known PRKD1 activators (e.g., PMA, oxidative stress inducers)

  • Inhibition: Include PRKD1 inhibitors as controls

  • Time course: Monitor changes in phosphorylation patterns over time

• Quantitative analysis approaches:

  • Measure co-localization coefficients between PRKD1 and phospho-substrates

  • Quantify nuclear/cytoplasmic ratios of PRKD1 and substrates

  • Use intensity correlation analysis to assess spatial relationships

• Advanced imaging techniques:

  • FRET-based reporters for PRKD1 activity

  • Live cell imaging with PRKD1-FITC antibody microinjection or cell-permeable versions

This methodological approach is particularly valuable for investigating how PRKD1 influences cell cycle progression through CDC25 phosphorylation, which represents a distinct mechanism from canonical checkpoint kinase pathways .

How should researchers interpret subcellular localization patterns of PRKD1 under different experimental conditions?

PRKD1 subcellular localization is dynamic and context-dependent, requiring careful interpretation:

• Normal baseline distribution:

  • PRKD1 primarily localizes to the cytoplasm and cell membrane under basal conditions

  • Some nuclear localization may be observed in specific cell types

• Stimulus-induced relocalization patterns:

  • DAG production at cell membranes recruits PRKD1, increasing membrane localization

  • Oxidative stress can induce nuclear accumulation of PRKD1

  • Cell cycle stage influences distribution pattern, particularly during G1 arrest

• Interpreting abnormal patterns:

  • Diffuse cytoplasmic staining with loss of membrane association may indicate pathway dysregulation

  • Nuclear accumulation outside of specific stimuli may suggest altered function

  • Punctate cytoplasmic pattern might represent vesicular association related to trafficking functions

• Quantitative assessment approaches:

  • Calculate nuclear/cytoplasmic intensity ratios across conditions

  • Measure membrane/cytoplasm intensity ratios

  • Perform line scan analysis across cellular compartments

• Colocalization interpretation:

  • Colocalization with membrane markers (e.g., Na+/K+-ATPase) indicates activation

  • Colocalization with nuclear transcription factors suggests active signaling

  • Colocalization with TLR9 or β-catenin indicates pathway-specific engagement

When interpreting PRKD1 localization patterns, it's essential to consider the cellular context, stimulation conditions, and potential impact of experimental manipulations on trafficking machinery.

What are common artifacts in PRKD1 antibody staining and how can they be distinguished from true signals?

Distinguishing authentic PRKD1 signals from artifacts requires awareness of common issues:

• Autofluorescence artifacts:

  • Appearance: Broad-spectrum emission not limited to FITC channel

  • Distinction: Present in unstained controls; often associated with fixatives like glutaraldehyde

  • Solution: Use appropriate autofluorescence quenching reagents; adjust acquisition settings

• Non-specific binding artifacts:

  • Appearance: Diffuse or punctate staining present in isotype controls

  • Distinction: Not reduced by PRKD1 knockdown; different pattern from expected subcellular localization

  • Solution: Optimize blocking (increase time/concentration); titrate antibody concentration

• Fixation artifacts:

  • Appearance: Altered localization pattern depending on fixation method

  • Distinction: Different patterns with different fixatives (compare PFA vs. ethanol fixation)

  • Solution: Validate with multiple fixation methods; compare to live cell studies when possible

• FITC photobleaching artifacts:

  • Appearance: Rapid signal loss during imaging; regional variation in intensity

  • Distinction: Progressive loss of signal with exposure; not seen in initial images

  • Solution: Use anti-fade mounting media; minimize exposure times; acquire images rapidly

• Cross-reactivity artifacts:

  • Appearance: Signals in cells/tissues not expected to express PRKD1

  • Distinction: Not eliminated by PRKD1 knockdown; may represent PRKD2/PRKD3 detection

  • Solution: Validate with recombinant monoclonal antibodies with confirmed specificity

For FITC-conjugated PRKD1 antibodies specifically, always include proper controls and validate staining patterns across multiple cell types with known PRKD1 expression profiles like HeLa and LNCaP cells .