PIP5K1C Antibody

What is PIP5K1C and what are its primary cellular functions?

PIP5K1C is a lipid kinase that catalyzes the production of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2/PIP2), a critical lipid second messenger that regulates multiple cellular processes. PIP5K1C demonstrates widespread expression in tissues including brain and skeletal muscle . Its primary functions include:

• Regulation of signal transduction pathways

• Membrane trafficking and vesicle dynamics

• Actin cytoskeleton remodeling and dynamics

• Cell adhesion and motility

• Focal adhesion formation and dynamics

• Synaptic vesicle transport and endocytosis

• Clathrin-coated pit assembly at synapses

Research has demonstrated that PIP5K1C also modulates the targeting of talins (TLN1 and TLN2) to the plasma membrane and their efficient assembly into focal adhesions . In pain signaling, PIP5K1C generates at least half of all PIP2 in dorsal root ganglia (DRG), including in small-to-medium diameter nociceptive DRG neurons .

What applications are PIP5K1C antibodies validated for?

PIP5K1C antibodies have been validated for multiple research applications based on the search results:

For optimal results, researchers should validate dilutions in their specific experimental systems, as the actual working concentration may vary depending on the sample type and detection method .

What species reactivity do PIP5K1C antibodies demonstrate?

The species reactivity of PIP5K1C antibodies varies by product. Based on the search results:

When selecting an antibody for cross-species studies, researchers should verify the validation data for each specific species of interest .

How should PIP5K1C antibodies be stored and handled to maintain optimal activity?

Based on the manufacturer recommendations across multiple suppliers:

• Store at -20°C for long-term storage (typically stable for one year after shipment)

• For frequent use and short-term storage, keep at 4°C for up to one month

• Avoid repeated freeze-thaw cycles as this may compromise antibody activity and specificity

• Most PIP5K1C antibodies are supplied in a storage buffer containing:

  • PBS with 0.02% sodium azide

  • 50% glycerol as a cryoprotectant

  • Sometimes with 0.5% BSA as a stabilizer

The storage conditions may affect antibody performance in different applications. For Proteintech antibody (27640-1-AP), the manufacturer notes that "aliquoting is unnecessary for -20°C storage" for 20μl sizes that contain 0.1% BSA .

What is the optimal protocol for using PIP5K1C antibodies in Western blotting?

For optimal Western blot detection of PIP5K1C, the following methodological considerations should be addressed:

Sample Preparation:

• Use appropriate lysis buffer containing phosphatase and protease inhibitors

• Load 20-50 μg of total protein per lane (may vary based on expression level)

Antibody Dilutions and Conditions:

• Primary antibody: 1:500-1:2000 dilution range (optimize for each antibody)

• Cell Signaling #3296 recommended dilution: 1:1000

• Proteintech 27640-1-AP recommended dilution: 1:500-1:1000

• Incubation: Typically overnight at 4°C or 2 hours at room temperature

Detection Considerations:

• Expected molecular weight varies by antibody and cell type:

  • Cell Signaling #3296: 87, 90 kDa

  • Proteintech 27640-1-AP: 70 kDa (observed) vs 73 kDa (calculated)

  • Boster A05687: 39 kDa (observed) vs 73260 MW (calculated)

  • Boster A05687-1: 111 kDa (observed) vs 73260 MW (calculated)

The variation in observed molecular weights across different antibodies suggests potential post-translational modifications, splice variants, or differences in gel migration conditions. Researchers should validate the expected band size in their specific experimental system .

How can I validate the specificity of my PIP5K1C antibody?

Validating antibody specificity is crucial for reliable research outcomes. Consider these approaches:

• Genetic models:

  • Use PIP5K1C knockout (KO) tissues/cells as negative controls

  • The research by Zylka et al. demonstrated complete absence of PIP5K1C staining in Pip5k1c−/− DRG embryonic mice, confirming antibody specificity

• Knockdown experiments:

  • Use siRNA/shRNA against PIP5K1C

  • Research has used Pip5k1c siRNA in ATDC5 cells to validate antibody specificity

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide

  • Several manufacturers offer blocking peptides for competition assays

• Multiple antibody validation:

  • Use different antibodies targeting distinct epitopes of PIP5K1C

  • Compare staining/blotting patterns across antibodies

• Expression correlation:

  • Compare protein levels with mRNA expression data

  • Verify subcellular localization matches known distribution patterns

The specificity of an antibody like Cell Signaling #3296 was confirmed in research where "No PIP5K1C protein was detectable in DRG from Pip5k1c−/− embryonic mice, confirming complete gene knockout and that both antibodies used in our study specifically recognized PIP5K1C" .

How can PIP5K1C antibodies be used to study its role in pain signaling pathways?

PIP5K1C plays a critical role in pain signaling pathways as demonstrated by research from Zylka and colleagues. Methodological approaches include:

• Expression analysis in nociceptive neurons:

  • PIP5K1C antibodies showed high colocalization with pain markers:

    • 98.1% colocalization with PAP (n=2,672 counted)

    • 97.5% colocalization with CGRP (n=2,094 counted)

    • 92.2% colocalization with TRPV1 (n=1,915 counted)

• Quantification of PIP2 levels in wild-type vs. PIP5K1C-deficient models:

  • Immunofluorescence staining revealed that membrane PIP2 levels were reduced by ~50% in small-to-medium diameter neurons of Pip5k1c+/− mice

  • PIP2 was reduced by ~70% in DRG from Pip5k1c−/− embryonic mice

• Functional calcium imaging to assess signaling deficits:

  • Researchers found impaired LPA and 17-PT PGE2 calcium signaling in Pip5k1c+/− neurons

  • Signaling deficits could be restored by delivering excess PIP2 to Pip5k1c+/− neurons before imaging

When designing experiments to study PIP5K1C in pain signaling, controls should include validation of antibody specificity in neuronal tissues and comparison with established pain markers through co-localization studies.

What role does PIP5K1C play in osteoarthritis, and how can antibodies help study this connection?

Recent research by Liu et al. (2023) has established a critical connection between PIP5K1C and osteoarthritis (OA). Experimental approaches using PIP5K1C antibodies revealed:

• Chondrocyte-specific deletion models:

  • Inducible deletion of Pip5k1c in aggrecan-expressing chondrocytes (cKO) caused multiple spontaneous OA-like lesions in aged (15-month-old) mice, but not in adult (7-month-old) mice

  • Lesions included cartilage degradation, surface fissures, subchondral sclerosis, meniscus deformation, synovial hyperplasia, and osteophyte formation

• Quantification of PIP5K1C expression in cartilage:

  • Immunofluorescent (IF) staining confirmed deletion of Pip5k1c in chondrocytes

  • Quantitative analyses showed percentages of Pip5k1c-positive cells were decreased by 26.7% in articular cartilage and 27.7% in growth plate in cKO mice compared to controls (P<0.0001)

• ECM protein expression analysis:

  • IF staining showed that Pip5k1c loss significantly decreased expression of anabolic ECM proteins (aggrecan and Col2a1) in articular cartilage

  • Percentages of aggrecan- and Col2a1-positive cells were decreased by 30.3% and 37.7%, respectively, in cKO versus control cartilages

To study PIP5K1C in OA pathogenesis, researchers should consider age-dependent effects, as phenotypes were observed in aged but not adult mice, suggesting PIP5K1C is particularly important for maintaining cartilage homeostasis during aging.

How does PIP5K1C expression correlate with cancer development, and what experimental approaches are recommended?

Recent research has identified a connection between PIP5K1C deficiency and cancer development, particularly in relation to PIKFYVE-dependent cancers:

• Molecular mechanism studies:

  • PIP5K1C deficiency distinguishes PIKFYVE inhibitor-sensitive cancers

  • The research found that "WX8-sensitive cells were deficient in PIP5K1C, an enzyme required to convert PtdIns4P into PtdIns(4,5)P2"

• Experimental approaches for studying this connection:

  • Manipulate PIP5K1C levels and observe effects on cancer cell sensitivity:

    • "Inhibition or ablation of PIP5K1C in WX8-resistant cells converted them into sensitive cells"

    • "Over-expression of PIP5K1C in WX8-sensitive cells increased their resistance to WX8"

• Potential clinical applications:

  • PIP5K1C levels could serve as a biomarker for identifying PIKFYVE-dependent cancers

  • "This discovery suggests that PIKFYVE-dependent cancers could be identified clinically by low levels of PIP5K1C and treated with PIKFYVE inhibitors"

When studying PIP5K1C in cancer, researchers should consider combining expression analysis using validated antibodies with functional studies that manipulate PIP5K1C levels (knockdown, overexpression) to establish causative relationships.

What are common issues when working with PIP5K1C antibodies and how can they be resolved?

Based on the search results and general antibody troubleshooting knowledge:

For specific applications, optimization strategies include:

• For IHC: Test different antigen retrieval methods. Proteintech suggests "antigen retrieval with TE buffer pH 9.0; alternatively, antigen retrieval may be performed with citrate buffer pH 6.0"

• For IF: Permeabilization conditions may need adjustment depending on subcellular localization

How can I design experiments to study PIP5K1C's role in specific cellular processes?

When designing experiments to study PIP5K1C's role in cellular processes, consider these methodological approaches:

• For studying focal adhesions and cell migration:

  • PIP5K1C "modulates the targeting of talins (TLN1 and TLN2) to the plasma membrane and their efficient assembly into focal adhesions"

  • Experimental design should include:

    • Co-immunoprecipitation to detect PIP5K1C-talin interactions

    • Co-localization studies using IF with focal adhesion markers

    • Cell migration/invasion assays after PIP5K1C manipulation

• For studying vesicle trafficking and endocytosis:

  • PIP5K1C is "required for synaptic vesicle transport" and "controls the plasma membrane pool of PtdIns(4,5)P2 implicated in synaptic vesicle endocytosis and exocytosis"

  • Experimental approaches:

    • Endocytosis assays using labeled transferrin or dextran

    • Live-cell imaging with markers for clathrin-coated pits

    • Synaptic vesicle recycling assays in neurons

• For studying phosphoinositide signaling pathways:

  • PIP5K1C generates PtdIns(4,5)P2, which "can directly act as a second messenger or can be utilized as a precursor to generate other second messengers: inositol 1,4,5-trisphosphate (IP3), diacylglycerol (DAG) or phosphatidylinositol-3,4,5-trisphosphate"

  • Recommended approaches:

    • PIP2 quantification using PIP2 antibodies or biosensors

    • Calcium imaging to monitor downstream signaling events

    • Pharmacological manipulation of PIP2 levels combined with PIP5K1C modulation

Essential controls for such experiments include:

• Use of PIP5K1C knockout/knockdown cells

• Rescue experiments with wild-type and catalytically inactive PIP5K1C

• Parallel studies with related isoforms (PIP5K1A, PIP5K1B) to determine specificity

What are the considerations for using PIP5K1C antibodies in models of neurological disorders?

PIP5K1C plays critical roles in neuronal function, making it relevant for studying various neurological disorders:

• Expression analysis in neural tissues:

  • PIP5K1C is present "on the plasma membrane and in the cytoplasm of most DRG neurons"

  • For neuronal studies, ensure antibodies are validated in neural tissues with proper controls

• Methodological considerations for pain hypersensitivity studies:

  • PIP5K1C is implicated in "pain hypersensitivity, inflammation, and neural dysfunction"

  • Research shows PIP5K1C is highly colocalized with pain markers:

    • PAP (98.1%)

    • CGRP (97.5%)

    • TRPV1 (92.2%)

  • Experimental design should include behavioral pain assays correlated with PIP5K1C expression/activity

• Considerations for studying synaptic function:

  • PIP5K1C is "required for synaptic vesicle transport" and "clathrin-coated pits assembly at the synapse"

  • For synaptic studies:

    • Use subcellular fractionation to isolate synaptic fractions

    • Perform immunocytochemistry with synaptic markers

    • Consider electrophysiological recording techniques to correlate with PIP5K1C levels

• Genetic models:

  • "In humans, homozygous mutations in the [PIP5K1C gene]" are associated with neurological conditions

  • Consider using patient-derived samples or genetic models mimicking human mutations

When designing experiments for neurological disorders, it's important to note that PIP5K1C functions may be cell-type specific and context-dependent, requiring careful selection of appropriate neural models and controls.

What emerging applications of PIP5K1C antibodies show promise for translational research?

Based on recent findings, several promising translational research directions for PIP5K1C antibodies include:

• Biomarker development for PIKFYVE-dependent cancers:

  • Recent research suggests "PIKFYVE-dependent cancers could be identified clinically by low levels of PIP5K1C and treated with PIKFYVE inhibitors"

  • Potential applications include immunohistochemical screening of cancer tissues to identify patients likely to respond to PIKFYVE inhibitors

• Therapeutic target identification for osteoarthritis:

  • Research shows "inducible deletion of Pip5k1c in aggrecan-expressing chondrocytes (cKO) causes multiple spontaneous OA-like lesions"

  • PIP5K1C antibodies could help identify patients with altered PIP5K1C expression who might benefit from targeted therapies

• Pain management research:

  • Studies show PIP5K1C "generates at least half of all PIP2 in the DRG, including in small-to-medium diameter, presumably nociceptive, DRG neurons"

  • PIP5K1C antibodies could help identify specific neuronal populations for targeted pain interventions

• Cell adhesion and migration in cancer metastasis:

  • PIP5K1C "modulates the targeting of talins (TLN1 and TLN2) to the plasma membrane and their efficient assembly into focal adhesions"

  • This role in cell adhesion makes it relevant for studying cancer cell invasion and metastasis