PIP4K2C Antibody, Biotin conjugated

What is PIP4K2C and why is it significant in research?

PIP4K2C (phosphatidylinositol-5-phosphate 4-kinase type II gamma) is an enzyme involved in phosphoinositide signaling pathways, with a calculated molecular weight of 47 kDa (421 amino acids) . This protein catalyzes the conversion of phosphatidylinositol-5-phosphate to phosphatidylinositol-4,5-bisphosphate, a key precursor in phosphoinositide signaling . PIP4K2C plays critical roles in multiple cellular processes including 1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate biosynthesis, negative regulation of 1-phosphatidylinositol-4-phosphate 5-kinase activity, and positive regulation of autophagosome assembly . Recent research has established PIP4K2C as a significant regulator of mammalian target of rapamycin complex 1 (mTORC1) signaling, with knockout studies demonstrating enhanced inflammatory responses . This makes PIP4K2C antibodies valuable tools for investigating signaling pathways related to metabolism, inflammation, and autophagy.

What are the typical applications for PIP4K2C antibodies?

PIP4K2C antibodies have been validated for multiple experimental applications according to the technical data:

For biotin-conjugated formats, these antibodies are particularly valuable for detection systems utilizing streptavidin-based amplification methods, which can significantly enhance sensitivity in assays with low-abundance targets . When designing experiments, researchers should note that optimal dilutions are sample-dependent and should be determined empirically for each specific application.

Which species reactivity has been confirmed for PIP4K2C antibodies?

The available PIP4K2C antibodies demonstrate variable cross-reactivity profiles:

When selecting antibodies for cross-species studies, researchers should prioritize antibodies with experimentally validated reactivity across their species of interest . For biotin-conjugated formats, expect similar species cross-reactivity profiles as their unconjugated counterparts from the same clone.

How should biotin-conjugated PIP4K2C antibodies be incorporated into multiplexed immunoassays?

For multiplexed immunoassays, biotin-conjugated PIP4K2C antibodies offer significant advantages due to their compatibility with streptavidin detection systems. When designing multiplexed experiments:

• Pair with appropriate capture antibodies: Some manufacturers offer validated matched antibody pairs specifically validated for multiplex applications. For example, product MP50878-2 uses 60616-1-PBS as capture and 60615-4-PBS as detection antibodies in cytometric bead assays .

• Optimize blocking conditions: Use 1-5% BSA or alternative blocking agents that don't contain biotin to prevent non-specific binding and background.

• Implement proper controls: Include single-analyte controls alongside multiplexed samples to verify that detection specificity is maintained in the multiplex format.

• Consider signal separation: When coupling with other detection systems (fluorescent or chemiluminescent), ensure spectral separation to avoid signal overlap.

The conjugation-ready formats of certain PIP4K2C antibodies (such as 60615-4-PBS) make them ideally suited for customized multiplexed assay development, particularly for cytometric bead arrays and ELISAs .

What are the recommended protocols for sample preparation when using PIP4K2C antibodies?

Sample preparation protocols should be optimized based on the specific application and sample type:

For Western Blot applications:

• Lyse cells in RIPA buffer containing protease and phosphatase inhibitors

• Determine protein concentration using Bradford or BCA assay

• Load 20-50 μg total protein per lane

• Include reducing agent in sample buffer and heat at 95°C for 5 minutes

For Immunohistochemistry applications:

• For tissue sections, antigen retrieval with TE buffer (pH 9.0) is recommended

• Alternatively, citrate buffer (pH 6.0) can be used for antigen retrieval

• For liver cancer tissue specimens specifically, both retrieval methods have been validated successfully

For Flow Cytometry applications:

• Use standard fixation and permeabilization protocols

• A 1:50 dilution is recommended for PE-conjugated formats

• Protect from light during all handling steps to preserve fluorescence intensity

Each application may require further optimization based on the specific experimental conditions and target tissue/cell type.

How can researchers validate specificity of PIP4K2C antibodies in their experimental system?

Validating antibody specificity is critical for reliable experimental outcomes. For PIP4K2C antibodies, implement these validation approaches:

• Positive controls: Test against samples with known expression (HeLa cells, HepG2 cells, and PC-12 cells have been confirmed to express detectable levels of PIP4K2C) .

• Knockout/knockdown validation: Published studies have utilized PIP4K2C knockout models that can serve as negative controls. At least two publications have used the 17077-1-AP antibody specifically in KD/KO validation experiments .

• Molecular weight verification: The observed molecular weight of PIP4K2C is approximately 47 kDa, which should be confirmed on Western blots .

• Multiple antibody approach: Compare results using antibodies from different clones or manufacturers targeting distinct epitopes of PIP4K2C.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm signal specificity.

When reporting results, always include detailed information about the validation methods employed to enhance reproducibility across research laboratories.

How can PIP4K2C antibodies be utilized to investigate its role in mTORC1 signaling?

Recent research has established PIP4K2C as an important regulator of mTORC1 signaling pathways . To investigate this relationship:

• Co-immunoprecipitation studies: Use PIP4K2C antibodies to pull down protein complexes and probe for known mTORC1 components.

• Phosphorylation analysis: Monitor phosphorylation status of key mTORC1 downstream targets (S6K, 4E-BP1) in relation to PIP4K2C expression/inhibition.

• Subcellular localization: Employ immunofluorescence with biotin-conjugated PIP4K2C antibodies to visualize co-localization with mTORC1 components.

• Pharmacological approaches: Compare PIP4K2C localization/activity before and after rapamycin treatment to assess functional relationships.

Studies using Pip4k2c knockout mice have demonstrated hyperactivation of mTORC1 signaling in multiple tissues, suggesting PIP4K2C negatively regulates this pathway . This provides an excellent model system for studying the intersection of phosphoinositide signaling and mTORC1 regulation using PIP4K2C antibodies as detection tools.

What experimental approaches can be used to study PIP4K2C's role in inflammation?

The involvement of PIP4K2C in inflammatory processes has been demonstrated in knockout mouse models . Researchers can utilize PIP4K2C antibodies to investigate:

• T-cell activation profiles: Flow cytometry using PIP4K2C antibodies in conjunction with T-cell markers can help examine the relationship between PIP4K2C expression and T-cell activation states.

• Cytokine measurement: Multiplex assays incorporating biotin-conjugated PIP4K2C antibodies alongside cytokine detection can establish correlation between PIP4K2C expression/activity and inflammatory cytokine production.

• Tissue infiltration studies: Immunohistochemistry with PIP4K2C antibodies can help characterize immune cell infiltrates in various tissues.

Research has shown that Pip4k2c knockout mice display increased inflammation and T-cell activation with aging, including elevated levels of proinflammatory cytokines (IFNγ, IL-12, IL-2) . These phenotypes were partially reversed by rapamycin treatment, suggesting a mechanistic link between PIP4K2C, mTORC1 signaling, and inflammatory regulation that can be further investigated using appropriate antibody-based techniques.

How can PIP4K2C antibodies contribute to antiviral research?

Recent studies have identified PIP4K2C as a potential target for broad-spectrum antiviral development . Researchers utilizing PIP4K2C antibodies in this context should consider:

• Viral replication assays: Monitor PIP4K2C expression and localization during viral infection cycles using immunofluorescence.

• Phosphoinositide profiling: Combine antibody-based detection of PIP4K2C with lipidomic analysis to correlate protein expression/localization with phosphoinositide signatures during viral infection.

• Inhibitor studies: Use PIP4K2C antibodies to confirm target engagement of novel inhibitors like RMC-113, which has demonstrated antiviral activity against multiple RNA viruses including SARS-CoV-2 .

• Autophagy pathway analysis: Investigate the relationship between PIP4K2C, autophagic flux, and viral replication using dual-labeling approaches with autophagy markers.

Research has demonstrated that PIP4K2C inhibition can reverse autophagic flux impairment induced by viruses, suggesting a potential mechanism for antiviral activity . Biotin-conjugated antibodies may be particularly useful in multi-parameter studies examining the intersection of phosphoinositide signaling, autophagy regulation, and viral replication.

How can researchers troubleshoot non-specific binding or high background when using PIP4K2C antibodies?

When encountering high background or non-specific binding with PIP4K2C antibodies:

• Optimize blocking conditions: Increase blocking agent concentration (3-5% BSA or serum) and extend blocking time (1-2 hours at room temperature).

• Adjust antibody concentration: Titrate antibody dilutions more extensively; for example, with the polyclonal antibody 17077-1-AP, test dilutions from 1:1000 to 1:8000 for Western blot to identify optimal signal-to-noise ratio .

• Increase washing stringency: Add 0.1-0.3% Tween-20 to wash buffers and increase washing duration/frequency.

• For biotin-conjugated antibodies specifically: Pre-block endogenous biotin using avidin/biotin blocking kits, particularly in tissues known to have high endogenous biotin (liver, kidney, brain).

• For IHC applications: Optimize antigen retrieval conditions by comparing TE buffer (pH 9.0) versus citrate buffer (pH 6.0) as recommended in the technical documentation .

• Consider sample-dependent factors: Different cell lines or tissue types may require specific optimization approaches; the antibody has been successfully used with HeLa, HepG2, and PC-12 cell lines .

What are the critical storage and handling considerations for maintaining biotin-conjugated PIP4K2C antibody activity?

To maintain optimal activity of biotin-conjugated PIP4K2C antibodies:

• Storage temperature: Store at the recommended temperature (-20°C for most formats, -80°C for certain specialized formats) .

• Avoid freeze-thaw cycles: Aliquot antibodies upon first thaw to minimize repeated freeze-thaw cycles that can degrade activity.

• Light protection: For biotin conjugates (and especially for dual-labeled antibodies), protect from light during storage and handling to prevent photobleaching.

• Buffer considerations: Most PIP4K2C antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) . Maintain these conditions when diluting.

• Avoid contamination: Use sterile technique when handling antibodies to prevent microbial contamination.

• For biotin conjugates specifically: Avoid storage in solutions containing free biotin or high concentrations of proteins with endogenous biotin.

According to product specifications, properly stored PIP4K2C antibodies remain stable for one year after shipment when stored at -20°C . For the PE-conjugated formats, the specific instruction is not to freeze, highlighting the importance of following format-specific storage recommendations .

What controls should be included when using PIP4K2C antibodies in knockout or inhibition studies?

When designing experiments involving PIP4K2C knockouts or inhibition:

• Wild-type controls: Always include wild-type/untreated samples as positive controls for antibody reactivity.

• Knockout validation: For genetic knockout studies, verify the absence of PIP4K2C using antibodies targeting different epitopes than those potentially affected by the knockout strategy.

• Loading/housekeeping controls: Include appropriate loading controls (β-actin, GAPDH) to normalize expression data.

• Isotype controls: For flow cytometry or IHC applications, include matched isotype controls (rabbit IgG for 17077-1-AP, mouse IgG1 for 60615-4-PBS) .

• Inhibitor specificity controls: When using inhibitors like RMC-113, include controls that address potential off-target effects, particularly since this compound has demonstrated dual inhibition of PIP4K2C and PIKfyve .

• Rescue experiments: To confirm specificity of knockout phenotypes, include rescue conditions where wild-type PIP4K2C is re-expressed.

Published studies have successfully used PIP4K2C antibodies in knockout validation experiments, demonstrating their utility in confirming genetic manipulation . These approaches help ensure experimental observations are specifically related to PIP4K2C rather than technical artifacts or off-target effects.

How might PIP4K2C antibodies support investigation of phosphoinositide signaling networks?

Future research utilizing biotin-conjugated PIP4K2C antibodies could advance understanding of phosphoinositide signaling through:

• Proximity labeling approaches: Using biotin-conjugated antibodies in conjunction with proximity labeling techniques to identify novel interaction partners in specific subcellular compartments.

• Single-cell analysis: Applying biotin-conjugated antibodies in single-cell proteomic approaches to understand cell-to-cell variation in PIP4K2C expression and its correlation with phosphoinositide profiles.

• Super-resolution microscopy: Leveraging the specificity of biotin-streptavidin interactions for super-resolution imaging of PIP4K2C localization relative to other phosphoinositide-modifying enzymes.

• Systems biology integration: Combining antibody-based detection of PIP4K2C with lipidomic and transcriptomic data to build comprehensive models of phosphoinositide regulation.

The established role of PIP4K2C in converting phosphatidylinositol-5-phosphate to phosphatidylinositol-4,5-bisphosphate positions it as a critical node in phosphoinositide signaling networks . Advanced antibody-based approaches will be essential for unraveling the complex interplay between different phosphoinositide species and their regulatory enzymes.

What is the potential for PIP4K2C as a therapeutic target based on current research?

Evidence from multiple studies suggests PIP4K2C may represent a valuable therapeutic target:

• Autoimmune applications: Research has identified a SNP at the PIP4K2C locus (rs1678542) in human patients with autoimmunity that might cause decreased PI5P4Kγ expression and increased mTORC1 signaling . This suggests therapeutic potential in autoimmune conditions.

• Cancer immunotherapy: Studies suggest that agents inhibiting PIP4K2C function could enhance cancer immunotherapy, potentially by modulating T-cell responses .

• Antiviral development: Recent research has identified PIP4K2C inhibition as a potential broad-spectrum antiviral approach, with the compound RMC-113 showing efficacy against multiple RNA viruses including SARS-CoV-2 .

• Metabolic disorders: Given PIP4K2C's role in regulating mTORC1 signaling, which is central to metabolic control, there may be applications in metabolic disorders.

Biotin-conjugated antibodies will play critical roles in target validation, mechanism studies, and biomarker development as these therapeutic directions advance. High-affinity, specific antibodies will be essential tools for assessing target engagement and pathway modulation during drug development processes.