PI4KB1 Antibody

What is PI4KB and what cellular functions does it regulate?

PI4KB, also known as PI4KIIIBETA, NPIK, PI4K-BETA, PI4K92, PI4KBETA, or PtdIns 4-kinase beta, is a 91.4 kilodalton protein that plays a crucial role in phosphoinositide metabolism . The enzyme catalyzes the phosphorylation of phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P), which serves as a precursor for other phosphoinositides like PI(4,5)P2. PI4KB is involved in membrane trafficking, particularly at the Golgi complex, and contributes to maintaining phosphoinositide pools that regulate various cellular processes including vesicular transport, cytoskeletal organization, and signal transduction. Unlike its counterpart PI4KA, which primarily functions at the plasma membrane, PI4KB mainly operates at the Golgi apparatus and other intracellular membrane compartments .

What species reactivity can I expect from commercially available PI4KB antibodies?

Commercial PI4KB antibodies demonstrate varying levels of species cross-reactivity. Based on the comprehensive antibody product information, most PI4KB antibodies show reactivity to human samples, with many exhibiting cross-reactivity with mouse and rat orthologs . Some antibodies offer broader reactivity profiles including rabbit, bovine, dog, guinea pig, hamster, and zebrafish samples . When selecting a PI4KB antibody for your research, it is essential to verify the specific reactivity profile of each antibody and consider preliminary validation experiments if working with less common species. Cross-species reactivity often correlates with sequence conservation in the epitope region targeted by the antibody.

What are the major applications of PI4KB antibodies in research?

PI4KB antibodies support multiple research applications with Western blot (WB) being the most commonly validated technique . Additional validated applications include:

Selection of the appropriate antibody should be guided by your specific research application and experimental conditions .

How can PI4KB antibodies be used to investigate membrane trafficking dynamics?

For advanced membrane trafficking studies, PI4KB antibodies can be employed in multiplexed imaging approaches to visualize spatial and temporal dynamics of phosphoinositide metabolism. Begin with fixed-cell immunofluorescence to establish baseline localization patterns, using PI4KB antibodies alongside organelle markers (e.g., TGN46 for trans-Golgi network). For dynamic studies, combine live-cell imaging of fluorescently-tagged PI4KB with PI4P biosensors such as the P4M domain from Legionella.

Methodologically, super-resolution microscopy techniques (STED, STORM) provide superior spatial resolution for examining PI4KB's association with membrane microdomains. For temporal studies, implement FRAP (Fluorescence Recovery After Photobleaching) or photoactivatable probes to track enzyme mobility and substrate accessibility. Quantitative image analysis should include colocalization coefficients (Pearson's or Mander's), intensity correlation analysis, and object-based colocalization to distinguish true biological associations from random overlap. This multi-parametric approach allows researchers to dissect PI4KB's contributions to secretory pathway function and membrane homeostasis with unprecedented precision.

What is the relationship between PI4KB and viral replication, and how can antibodies help study this interaction?

While PI4KA is directly implicated in hepatitis C virus (HCV) replication, PI4KB plays a crucial role in the replication of enteroviruses that repurpose the endoplasmic reticulum-Golgi trafficking machinery . To investigate these interactions using PI4KB antibodies, researchers can implement several methodological approaches:

• Proximity-based protein interaction assays: Employ proximity ligation assays (PLA) with antibodies against PI4KB and viral proteins to visualize and quantify direct interactions in situ with single-molecule sensitivity.

• Temporal analysis of redistribution: During infection time-course experiments, use PI4KB antibodies in immunofluorescence to track the enzyme's redistribution to viral replication organelles (VROs).

• Immunoprecipitation-mass spectrometry (IP-MS): Utilize PI4KB antibodies for IP followed by MS analysis to identify the complete interactome during different stages of viral infection.

• Inhibitor studies with immunodetection: Combine PI4K inhibitors with immunodetection of PI4KB to determine how enzyme activity versus structural functions contribute to viral replication.

This multifaceted approach can distinguish between PI4KB's enzymatic contributions and potential scaffold functions in viral replication complexes, providing insights into host-pathogen interactions that could inform antiviral therapeutic strategies.

How do I distinguish between PI4KA and PI4KB in experimental systems?

Distinguishing between these related phosphatidylinositol 4-kinases requires careful experimental design and antibody selection. Both enzymes catalyze the same reaction but differ in subcellular localization, size, and inhibitor sensitivity. Implement the following methodological approach:

• Antibody specificity verification: Validate antibody specificity through knockout/knockdown controls. PI4KA is typically 240 kDa while PI4KB is 91.4 kDa, allowing clear distinction on Western blots .

• Subcellular localization analysis: Use immunofluorescence with validated antibodies to distinguish characteristic localization patterns—PI4KA associates primarily with ER and plasma membrane, while PI4KB localizes predominantly to the Golgi apparatus.

• Inhibitor profiling: Employ selective pharmacological inhibitors; wortmannin inhibits PI4KB at nanomolar concentrations while requiring micromolar concentrations for PI4KA effects.

• Functional complementation: In knockdown or knockout systems, express one isoform to determine functional rescue capabilities, indicating distinct or overlapping roles.

• Protein complex characterization: Immunoprecipitate each kinase separately and analyze binding partners through proteomics to identify unique interaction networks.

This systematic approach allows confident discrimination between these kinases, essential for accurately attributing phenotypes to specific PI4K activities in cellular processes.

What are the optimal conditions for Western blotting with PI4KB antibodies?

Achieving optimal Western blot results with PI4KB antibodies requires careful attention to sample preparation, electrophoresis conditions, and detection parameters. Based on compiled protocols across multiple antibody product citations, implement the following methodological approach:

• Sample preparation: Lyse cells in RIPA buffer supplemented with phosphatase inhibitors (particularly important for studying phosphorylation-dependent interactions) and protease inhibitors. For membrane-associated PI4KB analysis, include 1% Triton X-100 to ensure complete solubilization.

• Protein loading and separation: Load 20-30 μg of total protein per lane. Use 8% polyacrylamide gels to achieve optimal resolution around the 91.4 kDa molecular weight of PI4KB .

• Transfer conditions: Employ semi-dry transfer (25V for 30 minutes) or wet transfer (30V overnight at 4°C) to ensure complete transfer of this relatively large protein.

• Blocking parameters: Block with 5% non-fat dry milk in TBST (TBS with 0.1% Tween-20) for 1 hour at room temperature to minimize background signal.

• Antibody incubation: Dilute primary antibodies according to manufacturer recommendations (typically 1:1000 to 1:2000) in blocking buffer and incubate overnight at 4°C with gentle agitation .

• Detection optimization: Use high-sensitivity ECL substrates for HRP-conjugated secondary antibodies or fluorescently-labeled secondary antibodies for quantitative analysis.

• Controls: Always include positive controls (cells/tissues known to express PI4KB) and negative controls (PI4KB-knockdown cells or non-relevant tissue) to validate specificity.

This protocol maximizes signal-to-noise ratio while ensuring reproducible detection of PI4KB across experimental conditions.

How can I optimize immunoprecipitation protocols for studying PI4KB protein interactions?

Optimizing immunoprecipitation (IP) for PI4KB interaction studies requires careful consideration of buffer composition, antibody selection, and validation steps. Implement this methodological framework:

• Lysis buffer optimization: Use a gentle lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) supplemented with protease and phosphatase inhibitors to preserve native protein interactions. For weaker interactions, consider crosslinking with DSP (dithiobis(succinimidyl propionate)) before lysis.

• Antibody selection: Choose antibodies specifically validated for IP applications . Consider epitope accessibility in the native protein conformation. For rabbit polyclonal antibodies, pre-clear lysates with protein A beads to reduce non-specific binding.

• Pre-clearing strategy: Pre-clear cell lysates with protein A/G beads for 1 hour at 4°C to remove proteins that bind non-specifically to the beads.

• Antibody binding: Incubate 2-5 μg of antibody with 500-1000 μg of protein lysate overnight at 4°C with gentle rotation.

• Wash optimization: Perform 5 washes with decreasing salt concentrations (starting with 300 mM NaCl, ending with 150 mM NaCl) to remove non-specific interactions while preserving specific ones.

• Elution considerations: For downstream mass spectrometry, elute with glycine buffer (pH 2.5) and immediately neutralize. For Western blot validation, elute directly in SDS sample buffer.

• Validation controls: Always include an isotype control antibody IP and input samples (5-10% of starting material) to assess enrichment efficiency.

This optimized protocol enhances the specificity and sensitivity of PI4KB immunoprecipitation, enabling reliable characterization of its interactome under various experimental conditions.

What approaches can I use to validate PI4KB antibody specificity?

Rigorous validation of PI4KB antibody specificity is crucial for generating reliable research data. Implement a comprehensive validation strategy incorporating these methodological approaches:

• Genetic knockdown/knockout controls: Generate PI4KB knockdown (siRNA, shRNA) or knockout (CRISPR-Cas9) cell lines to confirm antibody specificity through disappearance of the target band in Western blots or immunofluorescence signal.

• Overexpression systems: Complement negative controls with overexpression of tagged PI4KB (e.g., FLAG-tagged or GFP-fused) to confirm overlapping detection patterns between antibody staining and tag-specific detection.

• Peptide competition assays: Pre-incubate the antibody with excess immunogenic peptide before application to samples, which should abolish specific signal if the antibody is truly target-specific.

• Cross-reactivity assessment: Test the antibody against related family members, particularly PI4KA, to ensure selective detection of PI4KB.

• Multiple antibody concordance: Validate results using at least two antibodies targeting different epitopes of PI4KB, as concordant results strongly support specificity.

• Species cross-reactivity verification: When testing across species, confirm reactivity by sequence alignment of the epitope region and validation in species-specific positive controls.

• Application-specific validation: For each application (WB, IP, IHC, IF), perform separate validation procedures as antibody performance can vary significantly between applications.

Why might I observe multiple bands when performing Western blot with PI4KB antibodies?

Multiple bands in PI4KB Western blots can arise from several biological and technical sources. Address this methodological challenge through systematic analysis:

• Isoform identification: PI4KB may exist in alternatively spliced variants. Analyze predicted molecular weights against observed band patterns and consider RT-PCR validation of expression profiles in your experimental system.

• Post-translational modifications: Phosphorylation, ubiquitination, or other modifications can create mobility shifts. Confirm by treating samples with appropriate enzymes (e.g., phosphatases for phosphorylation) before electrophoresis to observe band collapse.

• Proteolytic processing: PI4KB may undergo specific cleavage events. Mitigate by using fresh samples, maintaining samples at 4°C during preparation, and including multiple protease inhibitors in lysis buffers.

• Technical artifacts: Non-specific binding or sample degradation can produce spurious bands. Optimize blocking conditions (try 5% BSA instead of milk for phospho-specific antibodies), increase washing stringency, and ensure sample integrity through proper handling.

• Cross-reactivity: The antibody may recognize related proteins. Validate specificity using knockdown/knockout samples and peptide competition assays as described in section 3.3.

• Loading and transfer issues: Overloading can cause signal spreading. Optimize protein amount (20-30 μg recommended) and ensure complete transfer by staining membranes post-transfer with Ponceau S.

Systematic elimination of these potential sources will help identify whether additional bands represent biologically meaningful variants or technical artifacts requiring protocol optimization.

How can I quantitatively analyze PI4KB expression or activity changes in different experimental conditions?

Quantitative analysis of PI4KB expression or activity requires careful experimental design and appropriate analytical approaches. Implement this methodological framework:

• Western blot quantification: For expression analysis, use fluorescently-labeled secondary antibodies rather than chemiluminescence for wider linear dynamic range. Normalize PI4KB signal to multiple housekeeping proteins (e.g., GAPDH, β-actin, and tubulin) to account for loading variations. Analyze across at least three biological replicates using software like ImageJ or specialized platforms like Image Studio (LI-COR).

• Kinase activity assays: To directly measure PI4KB enzymatic activity, immunoprecipitate the enzyme using validated antibodies and conduct in vitro kinase assays using either:

  • Radioactive assays: Measure incorporation of [γ-32P]ATP into PI substrate

  • Mass spectrometry: Quantify conversion of PI to PI4P

  • ELISA-based methods: Use PI4P-specific antibodies to detect product formation

• Cellular PI4P measurements: For intact cell analysis, implement immunostaining with PI4P-specific antibodies or biosensors (e.g., FAPP1-PH domain) combined with high-content imaging to quantify PI4P pools in different cellular compartments.

• Statistical analysis: Apply appropriate statistical tests based on data distribution. For normally distributed data, use parametric tests (t-test for two conditions, ANOVA for multiple conditions). For non-normally distributed data, apply non-parametric alternatives (Mann-Whitney or Kruskal-Wallis).

• Data visualization: Present data using scatter plots showing individual data points alongside means and standard deviations rather than bar graphs alone, following current best practices in data transparency.

This comprehensive quantitative approach provides robust measurements of both PI4KB expression levels and functional activity across experimental conditions.

What are common pitfalls in immunofluorescence studies with PI4KB antibodies and how can they be avoided?

Immunofluorescence studies with PI4KB antibodies present several potential pitfalls that can be systematically addressed through careful methodology:

• Fixation artifacts: Different fixation methods can dramatically affect antibody accessibility and epitope preservation. Compare paraformaldehyde (4%, 10-15 minutes) versus methanol fixation (-20°C, 10 minutes) in parallel to determine optimal epitope presentation. For membrane-associated proteins like PI4KB, adding 0.1% glutaraldehyde can better preserve membrane structures.

• Permeabilization optimization: PI4KB's membrane association requires balanced permeabilization—sufficient for antibody access but not excessive to preserve membrane structure. Test mild (0.1% saponin), moderate (0.1% Triton X-100), and stronger (0.5% Triton X-100) permeabilization conditions to optimize signal-to-noise ratio.

• Non-specific binding: This creates false positives and high background. Implement stringent blocking (5% normal serum from secondary antibody species plus 1% BSA, 1 hour at room temperature) and include primary antibody controls (isotype-matched irrelevant antibodies).

• Autofluorescence interference: Cellular components can generate autofluorescence that masks specific signals. Quench autofluorescence using 50 mM NH₄Cl treatment post-fixation and select fluorophores with emission spectra distinct from autofluorescence wavelengths.

• Bleed-through in co-localization studies: When examining PI4KB co-localization with other markers, sequential rather than simultaneous scanning in confocal microscopy reduces spectral overlap artifacts. Include single-stained controls to set acquisition parameters.

• Quantification challenges: Subjective visual assessment of localization is unreliable. Implement quantitative co-localization analysis using Pearson's or Mander's coefficients and intensity correlation analysis across multiple cells (n>30) from at least three independent experiments.

By systematically addressing these technical challenges, researchers can generate reliable immunofluorescence data on PI4KB localization and dynamics.

How does PI4KB function differ from other phosphatidylinositol kinases in cellular signaling?

PI4KB belongs to the phosphatidylinositol 4-kinase family but exhibits distinct functional characteristics compared to other phosphoinositide kinases. The methodological approach to understanding these differences involves comparative biochemical and cell biological analyses:

• Subcellular localization: Unlike PI4KA (primarily at plasma membrane and ER), PI4KB predominantly localizes to the Golgi apparatus, contributing to distinct pools of PI4P . Validate this through co-localization studies with organelle markers using both wildtype and kinase-dead mutants to distinguish catalytic from structural roles.

• Substrate specificity and catalytic properties: PI4KB and PI4KA both generate PI4P but differ in regulation and catalytic efficiency. PI4KB has lower intrinsic activity but can be stimulated by small GTPases like Arf1 and Rab11. Quantify these differences through in vitro kinase assays comparing Km and Vmax parameters.

• Protein interaction networks: PI4KB interacts with distinct effector proteins compared to other PI kinases. Map these interactions through proximity labeling techniques (BioID, APEX) followed by mass spectrometry to identify unique interactors.

• Inhibitor sensitivity profiles: PI4KB shows distinct sensitivity to pharmacological inhibitors. For example, it is inhibited by low concentrations of wortmannin (IC50 ~50-100 nM) while PI4KA requires much higher concentrations. Use inhibitor profiling to dissect specific contributions to cellular processes.

• Genetic perturbation phenotypes: PI4KB knockout or knockdown produces phenotypes distinct from other PI kinases. In particular, PI4KB depletion affects Golgi morphology and secretory traffic but has less impact on plasma membrane phosphoinositide homeostasis compared to PI4KA depletion .

This systematic comparative approach illuminates the unique role of PI4KB in phosphoinositide signaling and membrane trafficking pathways.

What are the current challenges and future directions in PI4KB antibody-based research?

The field of PI4KB antibody-based research faces several methodological challenges while offering promising future directions:

Current Challenges:

• Temporal resolution limitations: Traditional antibody-based detection captures static snapshots, limiting our understanding of PI4KB dynamics. This constraint complicates studying rapid phosphoinositide turnover during signaling events or membrane trafficking.

• Antibody specificity across applications: While many antibodies work well in denatured applications like Western blotting, fewer maintain specificity in native-state applications like immunoprecipitation or immunofluorescence . Cross-validation across applications remains inconsistent in the field.

• Quantitative standardization: Absolute quantification of PI4KB levels remains challenging due to variable antibody affinities and detection sensitivities, limiting cross-study comparisons.

• Distinguishing catalytic from scaffolding functions: Current antibody approaches often cannot distinguish between PI4KB's enzymatic activity and potential structural roles in protein complexes.

Future Directions:

• Nanobody development: Single-domain antibodies against PI4KB could enable live-cell imaging of endogenous protein with minimal perturbation, overcoming limitations of traditional antibodies or fluorescent protein fusions.

• Activity-state specific antibodies: Developing antibodies that specifically recognize active versus inactive conformations of PI4KB would revolutionize our understanding of its regulation.

• Spatial proteomics integration: Combining PI4KB antibody-based proximity labeling with subcellular fractionation and proteomics will map compartment-specific interaction networks.

• Super-resolution microscopy application: Optimizing PI4KB antibodies for techniques like STORM or STED will provide nanoscale resolution of its distribution relative to membrane domains and interaction partners.

• Single-molecule studies: Adapting PI4KB antibodies for single-molecule tracking will reveal new insights into its mobility, clustering, and activation dynamics in living cells.

These advances will transform PI4KB research from descriptive studies to mechanistic understanding of its dynamic functions in health and disease contexts.