PRKD2 Antibody

What is PRKD2 and what cellular functions does it regulate?

PRKD2 (Protein Kinase D2) is a member of the protein kinase D serine/threonine protein kinase family. It functions as a critical component in signal transduction pathways induced by phorbol esters and the CCK b gastrin receptor. PRKD2 exhibits strong homology to the serine/threonine protein kinases PKD/PKC and PKCnu, particularly in the duplex zinc finger-like cysteine-rich motif, pleckstrin homology domain, and protein kinase domain . Recent research has uncovered PRKD2's novel function in regulating insulin secretion, identifying it as a potential therapeutic target for metabolic diseases . Studies using PRKD2-knockout mouse models have demonstrated that PRKD2 deficiency promotes β-cell insulin secretion by increasing the expression and activity of L-type Ca²⁺ channels and subsequently augmenting high glucose- and membrane depolarization-induced Ca²⁺ influx .

What are the key differences between total PRKD2 antibodies and phospho-specific PRKD2 antibodies?

Total PRKD2 antibodies (such as 11623-1-AP) recognize the protein regardless of its phosphorylation state, making them suitable for general expression analysis . Phospho-specific antibodies (like Anti-Phospho-PKD2 S876) recognize PRKD2 only when phosphorylated at specific residues, making them valuable for studying activation states within signaling pathways . For comprehensive signaling studies, researchers often need to use both types of antibodies to correlate total expression with activation levels.

What species reactivity is available for commercially validated PRKD2 antibodies?

Current PRKD2 antibodies demonstrate cross-reactivity across multiple mammalian species due to the high conservation of the protein sequence. Based on validated data:

• The Anti-Phospho-PKD2 (S876) antibody from Boster (catalog # A04056S876-1) reacts with Human, Mouse, and Rat PRKD2 .

• The Phospho-PRKD2 (Ser876) Polyclonal Antibody from Thermofisher (PA5-40221) is predicted to react with Mouse and Rat based on sequence homology, in addition to its validated Human reactivity .

• The Proteintech PRKD2 antibody (11623-1-AP) has been tested and confirmed to react with Human, Mouse, and Rat samples, with citations also indicating reactivity in Zebrafish models .

When working with other species, preliminary validation experiments are strongly recommended to confirm cross-reactivity.

What are the recommended dilutions and conditions for PRKD2 antibody applications?

These recommendations provide starting points for assay optimization. The actual working concentration varies and should be determined empirically for each specific experimental system. For phospho-specific antibodies like Anti-Phospho-PKD2 (S876), inclusion of phosphatase inhibitors in sample preparation is critical to preserve the phosphorylation state .

How can researchers validate the specificity of PRKD2 antibodies in their experimental systems?

A comprehensive validation approach for PRKD2 antibodies should include:

• Positive Controls: Use tissues/cells known to express PRKD2 (e.g., human brain tissue, HeLa cells, human kidney tissue for Western blot applications) .

• Knockout/Knockdown Validation: Compare antibody reactivity between wild-type samples and those with PRKD2 knockdown or knockout. Published literature using PRKD2-KO mouse models provides valuable reference data .

• Phosphatase Treatment: For phospho-specific antibodies, treat one sample set with lambda phosphatase prior to analysis to confirm phospho-specificity .

• Peptide Competition: Pre-incubate the antibody with the immunizing peptide before application to determine specificity. Blocking peptides can be purchased for some antibodies like the Boster Anti-Phospho-PKD2 (S876) .

• Molecular Weight Verification: Confirm detection at the expected molecular weight (PRKD2 calculated MW is ~97 kDa) .

A rigorous validation strategy combining multiple approaches provides the strongest evidence for antibody specificity and reliability in experimental applications.

For Western Blotting:

• Use RIPA or NP-40 lysis buffers with complete protease inhibitor cocktails

• Add phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) when detecting phosphorylated PRKD2

• Sonicate briefly to shear DNA and reduce sample viscosity

• Heat samples at 95°C for 5 minutes in Laemmli buffer with reducing agent

For Immunohistochemistry:

• Fixation: 10% neutral buffered formalin is recommended

• Antigen retrieval: Use TE buffer pH 9.0 as primary method; citrate buffer pH 6.0 as alternative

• Blocking: 5-10% normal serum (species different from primary and secondary antibodies) with 1% BSA

• For phospho-PRKD2 detection, perfuse tissues with PBS containing phosphatase inhibitors before fixation

For Immunoprecipitation:

• Gentle lysis conditions (NP-40 or Triton X-100 based buffers)

• Pre-clear lysates with Protein A/G beads before adding PRKD2 antibody

• Recommended antibody amount: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Include validated positive controls (e.g., HeLa cells for human PRKD2)

How can PRKD2 antibodies be utilized to investigate its role in insulin secretion pathways?

Recent research has revealed PRKD2's crucial role in regulating insulin secretion, with PRKD2 deficiency triggering hyperinsulinemia which precedes insulin resistance and metabolic disorders . Researchers can implement the following experimental approaches:

• Co-immunoprecipitation Studies: Use PRKD2 antibodies to identify interaction partners within the insulin secretion pathway, particularly L-type Ca²⁺ channels which have been implicated in PRKD2-mediated regulation .

• Immunofluorescence Co-localization: Employ dual labeling with PRKD2 antibodies and markers of pancreatic β-cells to visualize PRKD2 distribution in islets under different metabolic conditions.

• Phosphorylation Dynamics: Monitor changes in PRKD2 Ser876 phosphorylation status in response to glucose stimulation using phospho-specific antibodies . This approach can reveal how PRKD2 activation correlates with insulin secretion events.

• Comparative Analysis in Disease Models: Compare PRKD2 expression and phosphorylation between normal and diabetic/insulin resistant models using both total and phospho-specific antibodies.

• Calcium Imaging Combined with Immunocytochemistry: Correlate PRKD2 localization with Ca²⁺ influx patterns in β-cells, as research indicates PRKD2 deficiency augments glucose-induced Ca²⁺ influx .

These methodologies can provide mechanistic insights into how PRKD2 regulates insulin secretion and potentially identify novel therapeutic targets for metabolic disorders.

What are the key phosphorylation sites of PRKD2 and their functional significance?

Serine 876 (S876) phosphorylation is particularly well-studied and commercially available antibodies specifically targeting this modification exist . This site serves as a reliable marker for PRKD2 activation status. Researchers should note that sample preparation for phosphorylation studies requires careful attention to phosphatase inhibitor inclusion during all steps of sample handling to preserve the physiological phosphorylation state.

When designing experiments to study PRKD2 phosphorylation dynamics, it is advisable to use both phospho-specific antibodies and total PRKD2 antibodies in parallel to normalize phosphorylation levels to total protein expression.

How do PRKD2 expression levels and functions differ across tissues in metabolic disease contexts?

Research using PRKD2 knockout models has revealed tissue-specific roles of PRKD2 in metabolic regulation:

• Pancreatic β-cells: PRKD2 deficiency increases insulin secretion by enhancing L-type Ca²⁺ channel expression and activity, leading to augmented glucose-induced Ca²⁺ influx .

• Liver: In PRKD2-deficient models, insulin-stimulated Akt ser473 phosphorylation is markedly decreased compared to wild-type mice, indicating development of insulin resistance .

• Skeletal Muscle: Similar to liver, PRKD2 knockout results in decreased insulin-stimulated Akt phosphorylation, contributing to systemic insulin resistance .

• Adipose Tissue: PRKD2 deficiency correlates with increased body weight, suggesting potential roles in adipogenesis or energy homeostasis .

For tissue-specific analysis, researchers should optimize antibody applications accordingly:

• For pancreatic tissue: Immunohistochemistry with antigen retrieval using TE buffer pH 9.0 is recommended for detecting PRKD2 in islets .

• For liver and muscle: Western blotting is effective for quantifying changes in both total PRKD2 and phospho-PRKD2 levels .

• For co-localization studies: Immunofluorescence approaches using PRKD2 antibodies alongside tissue-specific markers provide valuable spatial information.

How can researchers troubleshoot weak or inconsistent signals when using PRKD2 antibodies?

For phospho-specific PRKD2 antibodies specifically, researchers should note that phosphorylation states can rapidly change during sample handling. Quick processing of samples and consistent use of phosphatase inhibitors are critical for reproducible results with phospho-PRKD2 (S876) antibodies .

What are the optimal antigen retrieval methods for PRKD2 immunohistochemistry?

Based on validated protocols for PRKD2 antibodies, the following antigen retrieval methods are recommended:

• Primary Recommended Method: TE buffer (Tris-EDTA) at pH 9.0

  • Heat-induced epitope retrieval (HIER) using a pressure cooker or microwave

  • 20 minutes at sub-boiling temperature

  • Allow slow cooling to room temperature for 20 minutes before proceeding

• Alternative Method: Citrate buffer at pH 6.0

  • Heat to 95-100°C for 15-20 minutes

  • May be less effective than TE buffer for some tissue types but can be optimized

• For Phospho-specific Epitopes:

  • Add 1 mM sodium orthovanadate to the antigen retrieval buffer

  • Consider dual retrieval approach: citrate buffer followed by trypsin treatment

  • Validate each approach with known positive controls

The choice between methods should be experimentally determined for each specific tissue type and fixation protocol. For PRKD2 detection in pancreatic tissues, TE buffer pH 9.0 has shown superior results in detecting native protein while preserving tissue morphology .

How can researchers distinguish between PRKD2 and other PKD family members in experimental systems?

The protein kinase D family consists of three main members (PRKD1/PKD1, PRKD2/PKD2, and PRKD3/PKD3) with high sequence homology, particularly in their catalytic domains. To ensure specificity:

• Antibody Selection: Choose antibodies raised against unique regions of PRKD2. The immunogen information is critical - antibodies generated against the C-terminal region (AA range 829-878) like the Anti-Phospho-PKD2 (S876) are more likely to be specific for PRKD2 .

• Validation Strategy:

  • Test antibodies on overexpression systems of individual PKD family members

  • Validate with siRNA/shRNA knockdown specific to PRKD2

  • Use PRKD2 knockout models as negative controls

• Complementary Approaches:

  • Combine antibody-based detection with mRNA analysis (qPCR with isoform-specific primers)

  • Employ mass spectrometry for definitive identification of PRKD isoforms

  • Use multiple antibodies targeting different epitopes of PRKD2

• Molecular Weight Differentiation:

  • PRKD2: 97 kDa

  • PRKD1: 115 kDa

  • PRKD3: 110 kDa

  • Note that post-translational modifications can affect migration patterns

By implementing these strategies, researchers can confidently distinguish PRKD2 from other family members and avoid misinterpretation of experimental results.

How are PRKD2 antibodies being utilized to explore its potential as a therapeutic target for metabolic diseases?

Recent research has identified PRKD2 as a potential therapeutic target for metabolic diseases based on its role in insulin regulation . Researchers are utilizing PRKD2 antibodies in the following innovative approaches:

• Drug Discovery Screening: Phospho-specific antibodies against PRKD2 (S876) are being employed in high-throughput screening assays to identify compounds that modulate PRKD2 activity .

• Target Validation Studies: Combining PRKD2 knockout models with antibody-based detection methods to validate the relationship between PRKD2 deficiency and metabolic outcomes .

• Biomarker Development: Exploring phospho-PRKD2 levels as potential biomarkers for insulin resistance progression using quantitative immunoassays.

• Mechanism of Action Studies: Using co-immunoprecipitation with PRKD2 antibodies to identify interaction partners that could serve as alternative therapeutic targets in the same pathway.

• In vivo Efficacy Assessment: Employing immunohistochemistry with PRKD2 antibodies to evaluate tissue-specific responses to PRKD2-modulating compounds in preclinical models.

These approaches leverage the specificity of PRKD2 antibodies to advance understanding of its role in metabolic regulation and accelerate development of targeted therapeutics for conditions like insulin resistance and type 2 diabetes.

What are the latest findings regarding PRKD2's interaction with calcium channels in β-cells?

Recent research has revealed a critical relationship between PRKD2 and L-type Ca²⁺ channels in pancreatic β-cells that influences insulin secretion. Key findings include:

• PRKD2 deficiency promotes β-cell insulin secretion by increasing the expression and activity of L-type Ca²⁺ channels .

• In PRKD2 knockout models, there is augmented high glucose- and membrane depolarization-induced Ca²⁺ influx, directly linking PRKD2 to calcium signaling regulation .

• This enhanced calcium signaling leads to hyperinsulinemia, which precedes the development of insulin resistance and metabolic disorders in PRKD2-deficient models .

Researchers investigating these interactions can employ the following methodological approaches:

• Co-immunoprecipitation studies using PRKD2 antibodies to isolate and identify associated calcium channel components

• Proximity ligation assays to visualize PRKD2-calcium channel interactions in situ

• Phospho-proteomic analyses to identify PRKD2-mediated phosphorylation of calcium channel subunits

• Combined calcium imaging and immunocytochemistry to correlate PRKD2 localization with calcium influx patterns

Understanding this regulatory relationship provides new insights into the molecular mechanisms controlling insulin secretion and identifies potential intervention points for metabolic disease therapies.

What is the recommended storage and handling protocol for PRKD2 antibodies to maintain optimal performance?

Additional handling considerations:

• Freeze-Thaw Cycles: Minimize repeated freeze-thaw cycles as they can lead to antibody degradation and reduced performance. For antibodies used frequently, store a working aliquot at 4°C.

• Temperature Transitions: Allow refrigerated antibodies to equilibrate to room temperature before opening to prevent condensation which can introduce microbial contamination and accelerate degradation.

• Contamination Prevention: Use clean pipette tips and avoid touching the inside of the tube cap. Consider adding sterile BSA (0.1-1%) to diluted antibody solutions to enhance stability .

• Diluted Antibody Storage: If storing diluted working solutions, add preservatives (0.02% sodium azide) and protein carriers (1% BSA) and store at 4°C for short periods only (1-2 weeks).

• Documentation: Maintain a log of antibody use including freeze-thaw cycles and observed performance to track potential degradation over time.

Following these storage and handling guidelines will help maintain antibody integrity and ensure consistent experimental results when working with PRKD2 antibodies.

How can researchers effectively use PRKD2 knockout models to validate antibody specificity and biological findings?

PRKD2 knockout models serve as invaluable tools for antibody validation and biological discovery. Effective utilization includes:

• Antibody Validation Strategy:

  • Compare Western blot signals between wild-type and PRKD2-KO tissues/cells using the antibody in question

  • Include multiple tissue types as PRKD2 expression varies across tissues

  • Validate both total PRKD2 and phospho-specific antibodies independently

  • Document complete disappearance of the target band at the expected molecular weight (~97 kDa)

• Biological Finding Verification:

  • Establish baseline phenotypes in PRKD2-KO models (e.g., hyperinsulinemia, increased body weight)

  • Use immunohistochemistry to confirm altered expression of downstream targets (e.g., L-type Ca²⁺ channels)

  • Compare insulin-stimulated Akt ser473 phosphorylation between WT and PRKD2-KO in liver and skeletal muscle

  • Correlate glucose tolerance and insulin levels with PRKD2 expression status

• Experimental Controls:

  • Include heterozygous models to assess gene dosage effects

  • Consider tissue-specific knockout models to distinguish direct from secondary effects

  • Implement rescue experiments by reintroducing PRKD2 to confirm phenotype reversibility

Published research on PRKD2-KO mice has already established several key phenotypes, including hyperinsulinemia, increased body weight, and decreased insulin-stimulated Akt phosphorylation in liver and skeletal muscle tissue . These findings provide a valuable reference point for researchers validating their own experimental systems and antibodies.

How should researchers interpret variations in PRKD2 expression levels across different experimental models?

When analyzing PRKD2 expression data across experimental models, researchers should consider these interpretive frameworks:

• Baseline Variation Understanding:

  • Establish normal expression ranges in wild-type/control models using quantitative Western blotting with PRKD2 antibodies

  • Document tissue-specific expression patterns, as PRKD2 levels naturally vary between tissues

  • Consider developmental stage effects on expression, particularly in metabolic tissues

• Quantification Approaches:

  • Normalize PRKD2 to appropriate housekeeping proteins (β-actin, GAPDH)

  • For phospho-PRKD2, calculate phospho-to-total PRKD2 ratios rather than absolute phospho-PRKD2 levels

  • Use densitometry software for precise quantification of Western blot signals

  • Apply statistical analysis appropriate for sample size and distribution

• Biological Context Considerations:

  • Correlate PRKD2 expression changes with physiological parameters (insulin levels, glucose tolerance)

  • Assess compensation by other PKD family members in PRKD2-deficient models

  • Evaluate upstream regulators and downstream effectors in the same samples

  • Consider the kinetics of expression changes in response to experimental manipulations

• Common Misinterpretation Pitfalls:

  • Mistaking post-translational modifications for expression changes

  • Overlooking antibody cross-reactivity with other PKD family members

  • Assuming direct causality between PRKD2 expression and complex phenotypes

  • Generalizing findings from one tissue type to others without validation

What metrics should be used to evaluate the quality and reliability of PRKD2 antibody-based experimental results?

Additional quality considerations for specific applications:

For Western Blotting:

• Clean, distinct bands at the expected molecular weight (97 kDa for PRKD2)

• Linear relationship between protein amount and signal intensity within working range

• Consistent results when using different lysis buffers or sample preparation methods

For Immunohistochemistry:

• Specific cellular/subcellular localization consistent with known PRKD2 biology

• Appropriate positive controls (e.g., human colon cancer tissue)

• Minimal background staining in negative control sections

• Consistent staining pattern across technical replicates

For Phospho-specific Antibodies:

• Dynamic range of signal in response to known activators/inhibitors of the pathway

• Signal elimination following phosphatase treatment

• Correlation between phospho-signal and expected biological outcomes

Implementing these quality metrics ensures reliable interpretation of PRKD2 antibody-based experimental results and facilitates comparison across different studies and laboratories.

How can advanced imaging technologies enhance PRKD2 functional studies using antibody-based detection?

Emerging imaging technologies offer powerful new approaches for studying PRKD2 biology:

• Super-resolution Microscopy:

  • Techniques like STORM, PALM, and SIM overcome the diffraction limit of conventional microscopy

  • Enable visualization of PRKD2 localization at nanoscale resolution

  • Can resolve PRKD2 interactions with calcium channels at the plasma membrane of β-cells

  • Requires highly specific PRKD2 antibodies optimized for immunofluorescence applications

• Live-cell Imaging Approaches:

  • Antibody fragments (Fabs) labeled with fluorescent dyes for live-cell PRKD2 tracking

  • FRET-based biosensors to monitor PRKD2 activation in real-time

  • Correlative light-electron microscopy (CLEM) to link PRKD2 localization with ultrastructural features

  • Calcium imaging combined with PRKD2 visualization to study functional relationships

• Multiplexed Imaging:

  • Simultaneous detection of PRKD2, phospho-PRKD2, and interaction partners

  • Cyclic immunofluorescence (CycIF) for detecting >20 proteins in the same sample

  • Mass cytometry imaging (IMC) for highly multiplexed protein detection without spectral overlap

  • Spatial transcriptomics combined with PRKD2 protein detection

• Quantitative Analysis Techniques:

  • Machine learning algorithms for automated detection and quantification of PRKD2 in complex tissues

  • 3D reconstruction of PRKD2 distribution in intact islets or other tissues

  • Tracking PRKD2 translocation in response to stimuli with high temporal resolution

These advanced imaging approaches, when combined with validated PRKD2 antibodies, provide unprecedented insights into the dynamics of PRKD2 function in health and disease states, particularly in the context of insulin secretion and metabolic regulation.

What role might PRKD2 play in emerging therapeutic approaches for metabolic disorders?

PRKD2's newly discovered role in regulating insulin secretion positions it as a promising therapeutic target for metabolic disorders . Current research directions include:

• Target Validation Approaches:

  • Tissue-specific PRKD2 modulation using conditional knockout models

  • Correlation of PRKD2 activity with disease progression in clinical samples

  • Phenotypic rescue studies using viral vector-mediated PRKD2 reexpression

• Therapeutic Modulation Strategies:

  • Small molecule inhibitors of PRKD2 kinase activity

  • Activators of PRKD2 to potentially reduce hyperinsulinemia

  • Peptide-based modulators targeting specific PRKD2 protein interactions

  • Antisense oligonucleotides for selective PRKD2 knockdown

• Combination Therapy Approaches:

  • PRKD2 modulators with established diabetes medications

  • Targeting both PRKD2 and L-type calcium channels to fine-tune insulin secretion

  • Tissue-selective delivery to minimize off-target effects

• Biomarker Development:

  • Phospho-PRKD2 status as a predictor of therapeutic response

  • Monitoring PRKD2 activity as a pharmacodynamic marker

  • Correlation of PRKD2 genetic variants with metabolic phenotypes