PI4KA2 Antibody

What are the primary functions of PI4K enzymes targeted by these antibodies?

PI4KA specifically plays a critical role in maintaining plasma membrane phosphoinositide pools, particularly during strong stimulation of receptors coupled to phospholipase C activation. Research has demonstrated that PI4KA is essential for hepatitis C virus replication, making it a potential therapeutic target .

How do PI4K2A and PI4KA differ in their cellular localization and function?

PI4K2A demonstrates complex subcellular localization patterns including:

• Cytoplasm

• Membrane rafts

• Cell projections (dendrites)

• Synaptic junctions (presynaptic cell membrane and synaptosomes)

• Mitochondria

• Endosomes

• Cytoplasmic vesicles

In neurons, PI4K2A is localized in the cell body and transported to neuronal projections. It shows highest expression in kidney, brain, heart, skeletal muscle, and placenta, with lower expression in colon, thymus, and other tissues .

PI4KA, in contrast, is more specifically associated with endoplasmic reticulum exit sites and is critical for plasma membrane phosphoinositide generation. It plays an essential role in maintaining plasma membrane PIP2 pools during strong receptor stimulation coupled to phospholipase C activation .

What are the recommended applications for PI4K kinase antibodies?

Based on manufacturer specifications and research applications, PI4K antibodies are validated for several techniques:

These antibodies are particularly valuable for studying phosphoinositide signaling pathways, membrane trafficking, and host-pathogen interactions .

How should researchers design validation experiments for PI4K antibodies in new cell types or tissues?

When validating PI4K antibodies in new experimental systems, researchers should implement a multi-tiered validation approach:

What methodological considerations are critical when using PI4K antibodies for studying protein-protein interactions?

When investigating PI4K protein interactions:

• Gentle lysis conditions: Use mild detergents to preserve protein complexes while ensuring sufficient solubilization of membrane-associated PI4K proteins.

• Co-immunoprecipitation optimization: For antibodies validated for immunoprecipitation (like PI4 Kinase Antibody #4902, 1:50 dilution), optimize buffer conditions to maintain interaction integrity .

• Crosslinking considerations: Consider reversible crosslinking approaches before cell lysis to capture transient interactions, especially for membrane-localized complexes.

• Domain-specific interactions: Since PI4K2A has multiple cellular localizations (cytoplasm, membrane, endosomes), design experiments to distinguish interactions specific to each compartment .

• Functional validation: Complement interaction studies with functional assays measuring PI4-kinase activity to confirm biological relevance of identified interactions.

How can researchers effectively measure PI4K enzymatic activity in conjunction with antibody-based detection?

A comprehensive approach to measuring PI4K activity while using antibodies includes:

• In vitro kinase assays: After immunoprecipitation with PI4K antibodies (like #4902), perform kinase assays with purified PI substrate and 32P-ATP, followed by lipid extraction and thin-layer chromatography to detect PI4P formation .

• Cellular PIP2 measurements: Since PI4KA is crucial for maintaining plasma membrane PIP2 pools during strong stimulation, researchers can measure PIP2 levels using specific PIP2 biosensors before and after pharmacological inhibition or genetic knockdown of PI4KA .

• Visualization techniques: Combine antibody-based detection (immunofluorescence at 1:100 dilution for #4902) with phosphoinositide biosensors to correlate enzyme localization with activity sites .

• Agonist stimulation protocols: Design experiments with receptor agonists coupled to phospholipase C activation (such as angiotensin II mentioned in the research) to assess PI4KA's role in maintaining phosphoinositide pools under stimulated conditions .

• Inhibitor studies: Incorporate specific PI4K inhibitors as controls, noting that pharmacological blockade of PI4KA in animal models has shown severe consequences, including sudden death correlating with drug-induced PIP2 depletion after agonist stimulation .

What are common pitfalls in Western blot analysis using PI4K antibodies and how can they be addressed?

Several challenges may arise when using PI4K antibodies in Western blotting:

• High molecular weight detection issues: PI4 Kinase has a molecular weight of approximately 230 kDa , which can present technical challenges:

  • Use lower percentage gels (6-8%) for better separation

  • Extend transfer time with reduced voltage for complete transfer of high-MW proteins

  • Consider adding SDS (0.1%) to transfer buffer to aid high-MW protein transfer

• Non-specific binding: When following recommended dilutions (1:1000 for #4902, 1:500-1:2000 for CAB16546), researchers may still encounter non-specific bands :

  • Increase blocking time (overnight at 4°C)

  • Use alternative blocking agents (5% BSA instead of milk for phospho-specific detection)

  • Perform more stringent washing steps

• Sample preparation considerations:

  • For membrane-associated PI4Ks, ensure complete solubilization using appropriate detergents

  • Include phosphatase inhibitors to preserve phosphorylation status

  • Prevent protein degradation by using fresh samples and maintaining cold conditions

• Isoform-specific detection:

  • Verify antibody specificity against different PI4K isoforms

  • Include positive controls expressing known PI4K isoforms

How should researchers interpret contradictory results between PI4K antibody detection and functional assays?

When facing discrepancies between antibody detection and functional data:

• Expression vs. activity discrepancies: PI4K may be detected by antibodies but functionally inactive due to post-translational modifications or inhibitory protein interactions. Complementary approaches include:

  • Phospho-specific antibodies to detect activation state

  • In vitro kinase assays following immunoprecipitation

  • Mass spectrometry to identify post-translational modifications

• Localization-dependent activity: Since PI4K2A localizes to multiple subcellular compartments , activity may be compartment-specific:

  • Employ fractionation techniques to isolate different cellular compartments

  • Use immunofluorescence with co-localization studies

  • Perform proximity ligation assays to confirm interaction with regulatory partners

• Species-specific variations: Consider species differences when interpreting results, as antibody reactivity may vary (PI4 Kinase Antibody #4902 reacts with human, mouse, and rat proteins) .

• Genetic vs. pharmacological approaches: Research shows important distinctions between genetic and pharmacological studies of PI4KA. Genetic inactivation of PI4KA leads to death due to severe intestinal necrosis, while pharmacological blockade causes sudden death correlated with PIP2 depletion after agonist stimulation .

What strategies can resolve inconsistent immunofluorescence results with PI4K antibodies?

For researchers experiencing variable immunofluorescence results:

• Fixation optimization:

  • Test multiple fixation methods (4% paraformaldehyde, methanol, or combination approaches)

  • Optimize fixation time and temperature based on epitope accessibility

  • Consider antigen retrieval methods for formalin-fixed samples

• Permeabilization considerations:

  • Since PI4K2A has multiple localizations including membrane and cytoplasmic, compare different permeabilization agents (0.1% Triton X-100, 0.1% saponin, 0.05% SDS)

  • Adjust permeabilization time to balance cellular access with epitope preservation

• Signal amplification approaches:

  • For weak signals, consider tyramide signal amplification

  • Use higher antibody concentrations than recommended (>1:100 for #4902) with extended incubation times

  • Employ secondary antibodies with stronger fluorophores or quantum dots

• Controls for specificity:

  • Include peptide competition controls using immunogen sequences

  • Use siRNA knockdown samples as negative controls

  • Apply multiple antibodies targeting different epitopes of the same protein

How can PI4K antibodies be utilized in viral pathogenesis research, particularly for hepatitis C virus studies?

PI4KA has been identified as an essential host factor for hepatitis C virus (HCV) replication, making PI4K antibodies valuable tools in this research area :

What are the implications of using PI4K antibodies in cancer research based on recent findings?

While the search results primarily focus on PI4K's role in viral replication, connection to cancer research can be made through several angles:

• Phosphoinositide signaling in cancer:

  • Use PI4K antibodies to assess expression and localization in cancer vs. normal tissues

  • Investigate correlation between PI4K expression/activity and cancer progression

  • Study interaction between PI4K and known oncogenic signaling pathways

• Therapeutic targeting considerations:

  • The research on PI4KA inhibitors shows that pharmacological blockade leads to severe consequences, suggesting caution for cancer therapeutic development

  • Evaluate PI4K as a biomarker for cancer progression using immunohistochemistry with validated antibodies (IHC-P application of OAAB17392)

• Methodological approach for cancer studies:

  • Use multiple PI4K antibodies targeting different epitopes to comprehensively profile expression in tumor samples

  • Combine with phosphoproteomic approaches to assess activation state

  • Correlate with functional assays of PI4K activity in patient-derived samples

How should researchers design experiments using PI4K antibodies to study neurodegenerative disorders?

PI4K2A shows high expression in brain tissue and localizes to neuronal cell bodies and projections, suggesting potential relevance to neurodegenerative research :

• Expression profiling in disease models:

  • Use Western blotting with PI4K antibodies (1:1000 dilution of #4902) to compare expression levels between healthy and diseased brain tissues

  • Perform immunohistochemistry to assess regional distribution changes in disease states

  • Investigate age-dependent changes in PI4K expression and activity

• Subcellular localization studies:

  • PI4K2A localizes to dendrites, synaptic junctions, and synaptosomes

  • Use immunofluorescence (1:100 dilution of #4902) to study potential redistribution in disease models

  • Perform co-localization studies with markers of neurodegeneration (tau, amyloid, α-synuclein)

• Functional pathway analysis:

  • Investigate the relationship between PI4K-generated phosphoinositides and neuronal function

  • Study the impact of disease-associated mutations on PI4K activity and localization

  • Examine potential neuroprotective strategies targeting PI4K pathways

How might new antibody technologies enhance PI4K research beyond traditional applications?

Emerging antibody technologies offer new possibilities for PI4K research:

What considerations are important when developing quantitative assays using PI4K antibodies?

For developing robust quantitative assays:

• Antibody validation for quantitative applications:

  • Determine linear dynamic range for each antibody in quantitative Western blots

  • Assess lot-to-lot variability to ensure consistent quantification

  • Validate antibody performance across multiple sample types and preparation methods

• Standard curve development:

  • Create recombinant protein standards covering the physiological range of PI4K expression

  • Develop spike-in controls for various sample matrices

  • Include internal reference standards for normalization

• Multiplex assay considerations:

  • Test antibody performance in multiplex formats (multiplexed Western blot, mass cytometry)

  • Evaluate potential cross-reactivity with other phosphoinositide kinases

  • Optimize detection systems to minimize channel crosstalk

• Automation compatibility:

  • Assess antibody performance in high-throughput automated systems

  • Develop standardized protocols for reproducible quantification

  • Implement quality control measures for large-scale studies