PIP4K2C Antibody

What is PIP4K2C and what are its main biological functions?

PIP4K2C is a lipid kinase that belongs to the PI-5-phosphate 4-kinase family, which converts PI5P to PI45P2. Beyond this catalytic function, PIP4K2C plays critical roles in membrane trafficking, vesicular dynamics, autophagy regulation, and immune system modulation . Unlike its family members PIP4K2A and PIP4K2B, PIP4K2C has unique functions in regulating immune responses, particularly in cancer contexts . PIP4K2C also governs multiple aspects of membrane trafficking through its ability to regulate PI45P2 membrane localization and clustering, activities that appear to be independent of its catalytic function .

At the molecular level, PIP4K2C's calculated molecular weight is 47 kDa (421 amino acids) . Recent studies have revealed its crucial involvement in viral infections, including SARS-CoV-2, where it participates in viral entry, RNA replication, and assembly/egress processes .

How should I select the appropriate PIP4K2C antibody for my research?

Selection should be based on:

• Target epitope specificity: Consider whether you need antibodies targeting specific domains (e.g., C-terminal region, amino acids 310-381 or 348-364)

• Host species compatibility: Ensure the antibody won't cross-react with other proteins in your experimental system. Available options include mouse monoclonal and rabbit polyclonal antibodies

• Application requirements: Different antibodies are validated for specific applications:

  • Western blotting: Mouse monoclonal antibody AA 310-381

  • ELISA: Multiple options including mouse monoclonal antibodies

  • Cytometric bead array: Matched antibody pairs like 60616-1-PBS (capture) and 60615-4-PBS (detection)

• Reactivity spectrum: Confirm the antibody reacts with your species of interest (human, mouse, rat)

For multiplexed assays requiring paired antibodies, consider validated pairs like those offered for cytometric bead arrays (MP50878-2 or MP50878-3) .

What are the typical applications for PIP4K2C antibodies in basic research?

PIP4K2C antibodies can be employed for:

• Protein detection and quantification:

  • Western blotting to assess expression levels in different tissues or under various conditions

  • ELISA for quantitative measurement

• Localization studies:

  • Immunohistochemistry to examine tissue distribution patterns

  • Immunofluorescence for subcellular localization analysis

• Protein interaction studies:

  • Immunoprecipitation to identify binding partners

  • Pull-down assays to confirm target engagement of inhibitors or degraders

• Multiplex analysis:

  • Cytometric bead arrays for simultaneous analysis of multiple proteins

  • Mass cytometry applications for high-dimensional analysis

• Validation of knockdown efficiency:

  • Confirming siRNA-mediated depletion in functional studies

How do I optimize antibody working conditions for PIP4K2C detection?

Methodological approach for optimization:

• Initial concentration titration:

  • For Western blotting: Begin with 1:500-1:2000 dilution ranges

  • For ELISA: Start with 1-5 μg/mL and adjust based on signal-to-background ratio

  • For immunohistochemistry: Test 1:100-1:500 dilutions

• Blocking optimization:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Optimize blocking time (1-3 hours)

  • Consider using specialized blockers for phosphoprotein detection

• Sample preparation considerations:

  • For membrane proteins: Use appropriate lysis buffers that maintain membrane protein integrity

  • Include phosphatase inhibitors when studying phosphorylation states

  • Consider using intact cells for surface epitopes and permeabilized cells for intracellular domains

• Validation controls:

  • Positive control: Samples with known PIP4K2C expression (based on literature)

  • Negative control: PIP4K2C knockdown samples or tissues from knockout mice

  • Isotype control: Non-specific IgG from the same host species

• Storage and handling:

  • Follow manufacturer recommendations (e.g., store at -80°C for certain antibodies)

  • Avoid repeated freeze-thaw cycles

  • Consider adding preservatives for diluted antibody solutions

How can I distinguish between PIP4K2C and other PIP4K2 family members in my experiments?

PIP4K2 family members (PIP4K2A, PIP4K2B, and PIP4K2C) share over 60% similarity but exhibit differences in enzymatic activity levels and subcellular distribution . To distinguish them:

• Antibody selection strategy:

  • Choose antibodies targeting unique regions not conserved across family members

  • Use epitopes in the C-terminal region (e.g., AA 348-364 for PIP4K2C)

  • Validate specificity using knockout or knockdown controls for each family member

• Functional discrimination:

  • PIP4K2A/B positively regulates autophagic flux, while PIP4K2C has opposing functions

  • PIP4K2C uniquely regulates immune responses in cancer contexts

  • PIP4K2C specifically binds to SARS-CoV-2 nonstructural protein 6 (NSP6)

• Expression pattern analysis:

  • Compare tissue-specific expression profiles

  • Examine subcellular localization differences using fluorescently-tagged constructs

  • Analyze relative expression levels across different cell types

• Knockout/knockdown validation:

  • Design siRNA targeting unique regions of each family member

  • Use RT-qPCR with primers specific to each isoform to confirm knockdown efficiency

  • Employ CRISPR-Cas9 technology for complete knockout studies

What are the current methodologies for studying PIP4K2C's role in viral infections, particularly SARS-CoV-2?

Recent research has uncovered PIP4K2C's involvement in SARS-CoV-2 infection . Key methodological approaches include:

• Target engagement and inhibition studies:

  • Clickable probe analogs (e.g., SRN2-002) for pull-down assays

  • Competitive binding assays with inhibitors like RMC-113

  • Induced fit docking for in silico modeling of inhibitor binding

• Functional assessment of viral life cycle stages:

  • Viral entry assays using pseudotyped viruses

  • RNA replication quantification via RT-qPCR

  • Assembly/egress measurements through plaque assays

  • Single-cycle growth curves with timed inhibitor addition

• Mechanistic investigations:

  • Proteomic analysis to identify virus-host protein interactions

  • Single-cell transcriptomics to characterize cellular responses

  • Advanced lipidomics to track phosphoinositide signature alterations

  • Co-immunoprecipitation to confirm binding to viral proteins like NSP6

• Autophagic flux measurements:

  • LC3-I to LC3-II conversion assessment by Western blotting

  • Autophagy flux reporter systems (GFP-LC3-RFP)

  • Transmission electron microscopy for autophagosome visualization

  • Degradation of selective autophagy substrates

• Model systems:

  • Human lung organoids for physiologically relevant testing

  • Cell lines with ACE2 expression (e.g., A549-ACE2, Calu-3)

  • Primary cell cultures from different species to assess cross-species effects

How can PIP4K2C antibodies be used to investigate autophagy regulation mechanisms?

PIP4K2C has been implicated in autophagy regulation, with its inhibition or knockdown affecting autophagic flux . Methodological applications include:

• Monitoring autophagy marker changes:

  • Western blotting for LC3-I/II conversion with and without PIP4K2C manipulation

  • p62/SQSTM1 accumulation as an indicator of impaired autophagy

  • Tracking mutant huntingtin protein (mHTT) clearance, which is regulated by PIP4K2C

• Co-localization studies:

  • Double immunofluorescence for PIP4K2C and autophagosome markers

  • Live-cell imaging of fluorescently-tagged PIP4K2C with autophagy proteins

  • Super-resolution microscopy to visualize membrane interactions

• Functional autophagy assays:

  • Long-lived protein degradation assays following PIP4K2C knockdown

  • Mitophagy-specific measurements using MitoTracker-based approaches

  • Selective substrate degradation monitoring

• Signaling pathway analysis:

  • mTORC1 activity assessment, as PIP4K2C positively regulates mTORC1

  • ULK1 phosphorylation status examination

  • AMPK activation measurements following PIP4K2C manipulation

• Genetic rescue experiments:

  • Complementation with wild-type vs. catalytically inactive PIP4K2C

  • Domain-specific mutants to identify regions critical for autophagy regulation

  • Chimeric constructs with other PIP4K2 family members

What approaches can be used to study the correlation between PIP4K2C expression and immunological responses?

Given PIP4K2C's role in immune regulation and the link between PIP4K2C SNP (rs1678542) and autoimmunity , several methodological approaches are valuable:

• Genetic association studies:

  • Genotyping for rs1678542 in patients with autoimmune conditions

  • Correlation analysis between genotype and PIP4K2C expression levels

  • eQTL (expression quantitative trait loci) analysis

• Immune cell phenotyping:

  • Flow cytometry to assess immune cell populations in wild-type vs. Pip4k2c −/− mice

  • Cytokine profiling in plasma samples from knockout models

  • T-cell activation and differentiation assays

• Signaling pathway investigation:

  • mTORC1 activity measurement, as it's regulated by PIP4K2C and controls T-cell differentiation

  • Phospho-flow cytometry for pathway activation analysis

  • Multiplex cytokine assays before and after PIP4K2C inhibition

• In vivo models:

  • Tumor growth studies in Pip4k2c −/− mice or after treatment with PIP4K2C degraders

  • Autoimmunity models in knockout or inhibitor-treated animals

  • Adoptive transfer experiments to isolate cell-specific effects

• Therapeutic potential assessment:

  • Cancer immunotherapy response following PIP4K2C inhibition

  • Combination studies with checkpoint inhibitors

  • Ex vivo assays with human PBMCs treated with PIP4K2C degraders

How can I validate the specificity of my PIP4K2C antibody?

Methodological approaches for antibody validation:

• Knockout/knockdown controls:

  • Test antibody in samples following siRNA-mediated PIP4K2C depletion

  • Use CRISPR-Cas9 generated knockout cell lines

  • Compare with tissue from Pip4k2c −/− mice

• Peptide competition assays:

  • Pre-incubate antibody with excess immunizing peptide

  • Parallel processing of competed and non-competed samples

  • Expect signal elimination in competed samples for specific antibodies

• Multiple antibody approach:

  • Compare results using antibodies targeting different epitopes (e.g., C-terminal vs. internal regions)

  • Confirm consistent expression patterns with monoclonal and polyclonal antibodies

• Recombinant protein testing:

  • Use purified recombinant PIP4K2C as a positive control

  • Test against related family members (PIP4K2A, PIP4K2B) to confirm specificity

• Mass spectrometry validation:

  • Immunoprecipitate with the antibody and perform mass spectrometry analysis

  • Confirm target identification and assess potential cross-reactivities

What are common pitfalls when working with PIP4K2C antibodies, and how can they be addressed?

Common challenges and their solutions:

• Cross-reactivity with related kinases:

  • Use antibodies targeting unique regions not conserved across family members

  • Include family member knockouts as controls

  • Perform careful titration to minimize non-specific binding

• Detecting low expression levels:

  • Implement signal amplification strategies (HRP-conjugated secondary antibodies)

  • Consider immunoprecipitation before Western blotting

  • Use high-sensitivity detection systems (chemiluminescent substrates)

• Phosphorylation-dependent epitope masking:

  • Test antibody performance with and without phosphatase treatment

  • Use multiple antibodies targeting different regions

  • Consider phospho-specific antibodies if phosphorylation status is relevant

• Post-translational modification interference:

  • Optimize sample preparation to preserve or remove modifications as needed

  • Use denaturation conditions that expose relevant epitopes

  • Consider modification-specific antibodies for comprehensive analysis

• Technical issues in application-specific contexts:

  • For immunohistochemistry: Optimize antigen retrieval methods

  • For flow cytometry: Careful fixation and permeabilization protocol selection

  • For ELISA: Thorough blocking and washing optimization

How can I optimize PIP4K2C antibody-based assays for high-throughput screening of inhibitors or modulators?

Methodological considerations for high-throughput applications:

• Assay miniaturization and automation:

  • Adapt protocols to 384- or 1536-well formats

  • Optimize reagent concentrations for minimal volumes

  • Implement automated liquid handling systems

• Readout optimization:

  • Select high signal-to-background ratio detection methods

  • Consider homogeneous (no-wash) assay formats

  • Implement multiplex readouts to increase information content

• ELISA-based screening approaches:

  • Develop sandwich ELISA using validated antibody pairs

  • Optimize coating, blocking, and detection conditions

  • Implement quality control metrics (Z'-factor, signal window)

• Cell-based assay considerations:

  • Select appropriate cell models (endogenous vs. overexpression)

  • Develop stable reporter cell lines

  • Optimize fixation and antibody incubation times

• Target engagement confirmation:

  • Implement cellular thermal shift assays (CETSA)

  • Develop competition assays with clickable probes

  • Consider bioluminescence resonance energy transfer (BRET) approaches

How are PIP4K2C antibodies being used to study the development of novel PIP4K2C-targeted therapeutics?

Antibody applications in therapeutic development:

• Target validation studies:

  • Confirming the role of PIP4K2C in disease models using antibody-based detection

  • Correlation of expression levels with disease severity or progression

  • Tissue and cellular distribution mapping to anticipate on-target effects

• Inhibitor screening and characterization:

  • Target engagement confirmation for novel inhibitors like RMC-113

  • Competition assays with clickable analogs (e.g., SRN2-002)

  • Evaluation of selectivity against related kinases

• Degrader development support:

  • Monitoring protein levels following treatment with bifunctional degraders like LRK-A

  • Confirming specificity using whole-cell proteomic analyses

  • Time-course studies to assess degradation kinetics

• Therapeutic response biomarkers:

  • Developing assays to monitor PIP4K2C levels in clinical samples

  • Correlation of target engagement with efficacy measures

  • Patient stratification based on expression levels

• Combination therapy strategies:

  • Investigating PIP4K2C inhibition with immune checkpoint inhibitors

  • Monitoring pathway modulation in response to combination treatments

  • Assessment of synergistic effects on autophagic flux

What are the methodological considerations for investigating PIP4K2C's role in cancer immunotherapy?

Recent studies suggest PIP4K2C inhibition could enhance cancer immunotherapy . Key methodological approaches include:

• Immune cell functional assays:

  • T cell activation studies following PIP4K2C inhibition

  • Dendritic cell antigen processing and presentation assessment

  • NK cell cytotoxicity measurements with and without PIP4K2C modulation

• Tumor microenvironment analysis:

  • Multiplex immunohistochemistry to assess immune infiltration

  • Single-cell RNA sequencing of tumor and immune compartments

  • Spatial transcriptomics to map PIP4K2C expression patterns

• In vivo model systems:

  • Syngeneic tumor models in wild-type vs. Pip4k2c −/− mice

  • Treatment studies with PIP4K2C degraders showing tumor growth reduction and regressions

  • Humanized mouse models for human-specific effects

• Mechanism delineation approaches:

  • Pathway analysis focusing on mTORC1 signaling

  • Autophagic flux measurements in immune cells

  • Membrane trafficking assessment in antigen-presenting cells

• Translational considerations:

  • Development of companion diagnostics for patient selection

  • PIP4K2C SNP (rs1678542) genotyping correlation with treatment response

  • Biomarker studies in clinical trial samples

How can we investigate the relationship between PIP4K2C and other signaling pathways, particularly mTORC1?

Studies have linked PIP4K2C to mTORC1 signaling regulation . Methodological approaches include:

• Biochemical pathway analysis:

  • Phosphorylation status of mTORC1 substrates (S6K1, 4E-BP1) following PIP4K2C modulation

  • Co-immunoprecipitation studies to identify physical interactions

  • In vitro kinase assays to assess direct effects

• Genetic epistasis experiments:

  • Combined knockdown of PIP4K2C and mTOR pathway components

  • Rescue experiments with constitutively active pathway members

  • CRISPR screens to identify synthetic interactions

• Cellular assays:

  • Amino acid sensing studies, as mTORC1 is nutrient-responsive

  • Lysosomal positioning and recruitment of mTORC1 components

  • Autophagy induction measurements under various nutrient conditions

• Systems biology approaches:

  • Phosphoproteomic analysis following PIP4K2C inhibition

  • Network modeling of signaling pathway interactions

  • Integration with transcriptomic and metabolomic data

• Model organism studies:

  • Phenotypic comparison between Pip4k2c −/− mice and mTORC1 pathway mutants

  • Double mutant analysis for genetic interaction confirmation

  • Tissue-specific conditional knockouts to isolate context-dependent effects

What are the considerations for developing multiplex assays using PIP4K2C antibodies?

Several antibody pairs have been validated for multiplex applications . Key methodological approaches:

• Antibody pair selection:

  • Choose validated pairs like those listed for cytometric bead arrays (e.g., MP50878-2, MP50878-3)

  • Ensure epitopes don't overlap or interfere with each other

  • Test for cross-reactivity with other targets in the multiplex panel

• Conjugation strategies:

  • Use antibodies in conjugation-ready formats (BSA and azide-free)

  • Select compatible fluorophores with minimal spectral overlap

  • Consider site-specific conjugation methods for optimal performance

• Assay development considerations:

  • Optimize antibody concentrations individually before multiplexing

  • Validate specificity in the multiplex context with appropriate controls

  • Develop calibration curves for each target in the multiplex format

• Technical platforms:

  • Cytometric bead array systems for solution-phase multiplexing

  • Multiplex imaging platforms for spatial context preservation

  • Mass cytometry for high-dimensional analyses

• Data analysis approaches:

  • Implement compensation algorithms for spectral overlap

  • Develop appropriate normalization strategies

  • Apply machine learning for pattern recognition in complex datasets

How can PIP4K2C antibodies be adapted for advanced imaging techniques?

Methodological considerations for imaging applications:

• Super-resolution microscopy:

  • Select bright, photostable fluorophores for techniques like STORM or PALM

  • Consider direct conjugation to minimize distance between fluorophore and target

  • Optimize sample preparation to reduce background autofluorescence

• Live-cell imaging approaches:

  • Develop membrane-permeable nanobody derivatives

  • Consider genetically encoded tags with complementary fluorophores

  • Optimize imaging conditions to minimize phototoxicity

• Multiplex imaging strategies:

  • Sequential immunofluorescence with antibody stripping

  • Spectral unmixing for simultaneous detection of multiple targets

  • Mass cytometry imaging for highly multiplexed analyses

• Correlation with functional readouts:

  • Combine with activity sensors for phosphoinositides

  • Integrate with autophagic flux reporters

  • Correlate with calcium or pH indicators for functional contexts

• Tissue and 3D model applications:

  • Optimize clearing methods for thick specimens

  • Adapt antibody penetration protocols for organoids

  • Implement light-sheet microscopy for rapid 3D imaging

This comprehensive FAQ collection provides researchers with both fundamental and advanced methodological guidance for working with PIP4K2C antibodies across various research contexts. The increasing recognition of PIP4K2C's roles in viral infection, cancer immunotherapy, and autoimmunity makes these applications particularly relevant to current research priorities.