PI4KG7 Antibody

What are PI4K antibodies and what types exist in current research?

PI4K (Phosphatidylinositol 4-kinase) antibodies are immunological reagents designed to target PI4K enzymes, which catalyze the phosphorylation of phosphatidylinositol to phosphatidylinositol 4-phosphate. These enzymes play crucial roles in membrane trafficking, signaling pathways, and organelle function. Currently, two major types of PI4K antibodies are commonly used in research: anti-PI4KA (targeting the alpha isoform) and anti-PI4KB (targeting the beta isoform). PI4KA acts on phosphatidylinositol (PtdIns) in the first committed step in producing the second messenger inositol-1,4,5-trisphosphate . PI4KB may regulate Golgi disintegration and reorganization processes . Antibodies against these targets are typically available as polyclonal or monoclonal variants, with various modifications and conjugations depending on the intended application.

What are the primary research applications for PI4K antibodies?

PI4K antibodies are utilized in multiple experimental techniques including:

These applications enable researchers to investigate PI4K protein expression, localization, interactions, and functions in various experimental models . The versatility of these antibodies makes them valuable tools for both basic research and translational studies in areas such as cell biology, neuroscience, and cancer research.

How do I select the appropriate PI4K antibody for my research?

When selecting a PI4K antibody, consider these critical factors:

• Target specificity: Determine whether you need antibodies against PI4KA or PI4KB based on your research question. PI4KA (230kDa) and PI4KB (90-100kDa) have distinct cellular localizations and functions.

• Host species and reactivity: Verify that the antibody will recognize your species of interest. For example, many PI4K antibodies show reactivity with human, mouse, and rat samples .

• Application compatibility: Ensure the antibody has been validated for your specific application (WB, IHC, IF, IP).

• Clonality: Polyclonal antibodies (like those with catalog numbers OAGA02947 and 13247-1-AP) offer broader epitope recognition, while monoclonal antibodies provide higher specificity for a single epitope .

• Epitope location: Some antibodies target specific regions (e.g., the "middle region" of PI4KA), which may affect their utility in certain applications .

Always review the validation data and published literature citing the specific antibody to confirm its suitability for your experimental system.

What are the optimal conditions for using PI4K antibodies in immunofluorescence studies?

For successful immunofluorescence studies with PI4K antibodies, consider these methodological recommendations:

• Fixation method: For PI4KA and PI4KB, 4% paraformaldehyde fixation for 15-20 minutes at room temperature typically preserves antigenicity while maintaining cellular structure.

• Permeabilization: Use 0.1-0.3% Triton X-100 for 5-10 minutes to allow antibody access to intracellular PI4K proteins without disrupting membrane integrity.

• Blocking: Employ 5-10% normal serum (from the same species as the secondary antibody) with 1% BSA to minimize non-specific binding.

• Antibody dilution: Start with recommended dilutions (1:200-1:800 for PI4KB antibodies) , and optimize based on signal-to-noise ratio.

• Incubation conditions: For primary antibodies, incubate overnight at 4°C; for secondary antibodies, 1-2 hours at room temperature typically yields optimal results.

• Controls: Always include a negative control (omitting primary antibody) and, if possible, cells with PI4K knockdown or knockout as specificity controls .

• Counterstaining: DAPI for nuclear visualization helps to contextualize PI4K localization patterns.

These parameters should be systematically optimized for your specific cell type and experimental question to achieve reproducible results.

How can I validate the specificity of my PI4K antibody?

Validating antibody specificity is critical for ensuring reliable results. Implement these validation approaches:

• Genetic knockdown/knockout: Compare staining in wild-type cells versus cells where PI4K has been depleted via RNAi or CRISPR/Cas9. The search results indicate this approach has been used successfully for PI4KB antibody validation in NIH-3T3 cells .

• Peptide competition: Pre-incubate the antibody with the immunizing peptide or recombinant protein (such as the PI4KA recombinant protein encompassing the middle region) before applying to your sample—specific signals should be blocked.

• Multiple antibodies: Test antibodies targeting different epitopes of the same protein—concordant results increase confidence in specificity.

• Cross-species reactivity: If the antibody is predicted to react with multiple species, consistent patterns across species support specificity.

• Molecular weight verification: In Western blots, confirm that the observed band matches the expected molecular weight (approximately 230 kDa for PI4KA, 90-100 kDa for PI4KB) .

• Subcellular localization: Verify that the detected localization aligns with known literature (e.g., PI4KB associates with the Golgi apparatus) .

Thorough validation using multiple approaches provides the strongest evidence for antibody specificity.

What experimental controls are essential when using PI4K antibodies?

Robust experimental design requires appropriate controls:

For immunohistochemistry specifically, include appropriate antigen retrieval controls—PI4KB antibody detection in human liver tissue has been optimized using TE buffer at pH 9.0 or citrate buffer at pH 6.0 .

How can PI4K antibodies be integrated into antibody-cell conjugation (ACC) technologies?

Antibody-cell conjugation (ACC) represents an innovative research direction for utilizing PI4K antibodies beyond conventional applications. In this approach, PI4K antibodies can be conjugated to immune cells to create novel therapeutic tools. Based on current ACC methodologies, PI4K antibodies could be integrated using these strategies:

• Metabolic sugar engineering approach: PI4K antibodies can be modified with DBCO-PEG4-NHS ester while target cells (e.g., NK-92 cells) are treated with azide moieties. The antibody-cell conjugation then occurs via bioorthogonal azide-alkyne click chemistry reaction .

• Chemoenzymatic methods: PI4K antibodies could be coupled to GDP-fucose and then transferred to cell surface glycocalyxes using H. pylori-derived α-1,3-fucosyltransferase. This method is rapid, biocompatible, and requires no genetic modification .

• NHS-DNA coupling approach: This three-step method involves coupling single-stranded DNA to PI4K antibodies, attaching complementary DNA to cell surface proteins, and then hybridizing these complementary strands to create the final conjugate .

These techniques could potentially enable PI4K antibodies to direct immune cells toward cells expressing PI4K target proteins, offering new research tools for studying PI4K functions in cellular contexts.

What are the challenges in using PI4K antibodies for studying membrane trafficking?

Researchers face several methodological challenges when using PI4K antibodies to study membrane dynamics:

• Fixation artifacts: PI4K proteins, particularly PI4KA, can relocalize during fixation, potentially leading to misinterpretation of their true localization. Comparing multiple fixation methods (paraformaldehyde, methanol, glutaraldehyde) is recommended to identify potential artifacts.

• Temporal resolution limitations: Traditional immunofluorescence provides only static snapshots of PI4K localization, limiting insights into dynamic trafficking processes. Complementary live-cell imaging with fluorescently tagged PI4K may be necessary.

• Epitope accessibility: PI4K proteins often function in multi-protein complexes where antibody epitopes may be masked. Optimizing antigen retrieval techniques (as shown for PI4KB in human liver tissue) becomes crucial.

• Distinguishing isoforms: Due to sequence homology between PI4K family members, ensuring antibody specificity is essential. Validation using RNAi approaches, as demonstrated for PI4KB , helps confirm isoform-specific detection.

• Quantification challenges: Accurately quantifying PI4K relocalization during trafficking events requires sophisticated image analysis methods and proper normalization to account for expression level variations.

Addressing these challenges requires combining multiple technical approaches and rigorous controls to validate findings.

How do PI4K antibody-based assays compare with functional enzyme activity assays?

Understanding the relationship between PI4K protein detection (via antibodies) and enzyme function requires considering these comparative aspects:

For comprehensive PI4K characterization, combining antibody detection methods (to determine "where" and "how much") with activity assays (to determine "how active") provides complementary insights. This integrated approach is particularly valuable when investigating how point mutations or post-translational modifications might affect PI4K function without altering total protein levels.

How do I address inconsistent results when using PI4K antibodies?

When facing inconsistent results with PI4K antibodies, implement this systematic troubleshooting approach:

• Antibody quality assessment:

  • Check antibody age and storage conditions—glycerol-preserved antibodies (like the PI4KB antibody containing 50% glycerol) should be stored at -20°C.

  • Avoid repeated freeze-thaw cycles by preparing small aliquots.

  • Verify that the antibody concentration remains within the effective range (e.g., 0.64 mg/ml for PI4KA antibody) .

• Protocol optimization:

  • Titrate antibody concentrations systematically—recommended dilution ranges vary by application (1:500-1:3000 for WB, 1:50-1:500 for IHC, 1:100-1:1000 for IF/ICC) .

  • Optimize blocking conditions to reduce background.

  • For IHC applications, test different antigen retrieval methods as suggested for PI4KB (TE buffer pH 9.0 or citrate buffer pH 6.0) .

• Sample preparation issues:

  • Ensure consistent sample harvesting, lysis, and processing.

  • Verify protein integrity with total protein stains before antibody probing.

  • Consider the impact of cell confluency and culture conditions on PI4K expression levels.

• Detection system variables:

  • Use fresh detection reagents (ECL for WB, fluorescent secondary antibodies for IF).

  • Standardize exposure times and imaging parameters across experiments.

Document all troubleshooting steps systematically to identify the variables affecting your results.

How can I resolve cross-reactivity issues with PI4K antibodies?

Cross-reactivity can compromise experimental interpretation. Address this issue through:

• Antibody selection refinement:

  • Choose antibodies validated against multiple PI4K family members.

  • Consider monoclonal antibodies when cross-reactivity is a persistent issue.

  • Review the immunogen sequence information—antibodies raised against unique regions (like the "middle region" of PI4KA) may offer higher specificity.

• Experimental validation strategies:

  • Perform parallel experiments with genetic knockdown models (as demonstrated for PI4KB in NIH-3T3 cells) .

  • Include blocking peptides specific to your target PI4K isoform.

  • Compare results across multiple antibodies targeting different epitopes of the same protein.

• Optimization techniques:

  • Increase washing stringency (longer washes, higher detergent concentration).

  • Use higher dilutions of primary antibody to reduce non-specific binding.

  • For WB applications, optimize transfer conditions based on the molecular weight of your specific PI4K isoform (approximately 230 kDa for PI4KA, 90-100 kDa for PI4KB) .

• Alternative approaches:

  • Consider using tagged recombinant PI4K versions where antibody specificity is problematic.

  • Employ mass spectrometry-based approaches for definitive protein identification.

These strategies can significantly improve signal specificity while reducing background issues.

What are the best practices for quantitative analysis of PI4K levels using antibody-based methods?

For reliable quantification of PI4K proteins using antibody-based techniques:

• Western blot quantification:

  • Always work within the linear dynamic range of detection.

  • Use internal loading controls appropriate for your experimental context.

  • Consider triplet biological replicates minimum for statistical analysis.

  • Normalize band intensity to total protein rather than single housekeeping proteins when possible.

  • For PI4KB Western blots, the observed molecular weight of approximately 100 kDa should be consistent across samples .

• Immunofluorescence quantification:

  • Acquire images using identical exposure settings across all samples.

  • Analyze raw, unprocessed images to avoid introducing artifacts.

  • Establish clear criteria for defining regions of interest.

  • Consider z-stack acquisition to account for the three-dimensional distribution of PI4K proteins.

  • Present data as relative fluorescence intensity rather than absolute values.

• Statistical considerations:

  • Apply appropriate statistical tests based on data distribution.

  • Report both biological and technical variability.

  • Consider power analysis to determine adequate sample sizes.

  • Be transparent about outlier identification and handling.

• Validation approaches:

  • Confirm antibody-based quantification with orthogonal methods (qPCR, mass spectrometry).

  • Validate findings across multiple cell lines or tissue types when possible.

These quantification approaches ensure that observed differences in PI4K levels reflect true biological variation rather than technical artifacts.

How can PI4K antibodies be applied in studying disease mechanisms?

PI4K antibodies are becoming increasingly valuable for investigating disease pathophysiology:

• Cancer research applications:

  • PI4K antibodies enable detection of altered expression patterns in tumor versus normal tissues.

  • Immunohistochemistry applications (using dilutions of 1:50-1:500) can reveal PI4K distribution changes during cancer progression.

  • Combined with patient outcome data, PI4K immunostaining might identify prognostic biomarkers.

• Infectious disease mechanisms:

  • Several viruses (including hepatitis C and enteroviruses) hijack PI4K enzymes for replication.

  • PI4K antibodies help visualize viral replication complexes and their colocalization with PI4K enzymes.

  • Understanding these interactions provides potential therapeutic targets.

• Neurodegenerative disorders:

  • Phosphoinositide signaling dysregulation has been implicated in various neurological conditions.

  • PI4K antibodies can reveal altered distribution in affected tissues.

  • Combining PI4K immunostaining with markers of neurodegeneration helps establish mechanistic links.

• Metabolic disease research:

  • PI4K enzymes influence insulin signaling and glucose metabolism.

  • Antibody-based approaches help track PI4K expression and localization changes in metabolic tissues.

As these research areas develop, validating findings across multiple antibodies and complementary techniques becomes increasingly important.

What novel techniques are emerging for PI4K antibody applications in research?

Several innovative methodologies are expanding the utility of PI4K antibodies:

• Proximity labeling approaches:

  • BioID or APEX2 fusion with PI4K enzymes combined with antibody validation identifies proximal interacting partners.

  • This approach reveals the dynamic PI4K interactome under different cellular conditions.

• Super-resolution microscopy integration:

  • Techniques like STORM and PALM, combined with highly specific PI4K antibodies, reveal nanoscale organization of PI4K enzymes.

  • These approaches require rigorous validation of antibody specificity to avoid artifacts.

• Antibody-based proteomics:

  • IP-mass spectrometry using validated PI4K antibodies (as demonstrated for PI4KB in K-562 cells) enables comprehensive analysis of PI4K-associated protein complexes.

  • This approach reveals context-dependent changes in the PI4K interactome.

• Live-cell applications via antibody fragments:

  • Developing cell-permeable antibody fragments targeting PI4K enzymes could enable dynamic tracking in living cells.

  • These approaches complement traditional fixed-cell immunostaining methods .

• Antibody-cell conjugation technologies:

  • Emerging ACC techniques using chemical coupling approaches (like NHS-DNA coupling or chemoenzymatic methods) could potentially incorporate PI4K antibodies.

  • Such conjugates might enable targeted delivery of payloads to cells with altered PI4K expression.

These emerging technologies represent the frontier of PI4K antibody applications in research, expanding beyond traditional detection methods to functional and therapeutic approaches.