ITPKC Antibody

What is ITPKC and what is its biological function?

ITPKC (Inositol-trisphosphate 3-kinase C), also known as IP3KC, belongs to the inositol phosphokinase (IPK) family . It functions primarily as an enzyme that phosphorylates inositol 2,4,5-triphosphate to inositol 2,4,5,6-tetraphosphate . This enzyme plays a crucial role in calcium signaling pathways and is relatively weakly activated by the calcium-calmodulin complex. More importantly, ITPKC acts as a negative regulator of T-cell activation through the Ca²⁺/NFAT signaling pathway . IP3 is generated through the hydrolysis of phosphatidylinositol 4,5-biphosphate by phospholipase C in response to various external stimuli . In T cells specifically, IP3 released by stimulation of the TCR complex increases intracellular Ca²⁺ through IP3 receptors expressed on the endoplasmic reticulum . This calcium influx ultimately leads to nuclear translocation of nuclear factor of activated T cells (NFAT) and activates transcription of interleukin-2 and other cytokines .

What are the key applications for ITPKC antibodies in research?

ITPKC antibodies serve multiple applications in research settings, with varying recommended dilutions for optimal results. The primary applications include:

These applications enable researchers to detect ITPKC expression in various experimental contexts, from protein quantification to cellular localization studies . When implementing these techniques, it is essential to titrate the antibody in each testing system to obtain optimal results, as the effectiveness can be sample-dependent .

What tissues and cell types show ITPKC expression?

In experimental validation, positive Western blot detection has been confirmed in MDA-MB-453s cells and human heart tissue . Immunohistochemistry has successfully detected ITPKC in human breast cancer tissue, human heart tissue, and human lymphoma tissue . For researchers studying ITPKC in cell culture models, HepG2 cells have been validated for both immunoprecipitation and immunofluorescence applications .

How should ITPKC antibodies be stored and handled?

Proper storage and handling of ITPKC antibodies are crucial for maintaining their reactivity and specificity. The recommended storage conditions are:

• Store at -20°C for long-term stability

• Most ITPKC antibodies remain stable for one year after shipment when stored properly

• For frequent use and short-term storage (up to one month), antibodies can be kept at 4°C

• Avoid repeated freeze-thaw cycles as this can degrade antibody quality

The typical storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Some preparations may contain additional stabilizers such as 0.1% BSA in smaller volume preparations (20μl sizes) . For antibodies in liquid form, no reconstitution is required before use . Always handle antibodies according to the manufacturer's instructions to ensure optimal performance in experimental applications.

What validation methods confirm ITPKC antibody specificity?

Confirming antibody specificity is essential for generating reliable research data. Several validation methods should be employed when working with ITPKC antibodies:

• Western blotting verification: ITPKC has a calculated molecular weight of 75 kDa, but the observed molecular weight is typically 100-105 kDa . This discrepancy is important to note when interpreting Western blot results.

• Positive and negative controls: Use known positive samples (such as MDA-MB-453s cells or human heart tissue) and appropriate negative controls .

• Cross-reactivity testing: Most commercially available ITPKC antibodies show reactivity with human samples, but cross-reactivity with other species should be verified if using non-human models .

• Blocking peptide controls: Some suppliers offer blocking peptides that can be used to confirm specificity in immunohistochemistry or Western blot applications .

• Multiple application validation: Reputable suppliers validate antibodies across multiple applications (WB, IHC, ICC, IF, ELISA) using known positive and negative samples .

Thorough validation ensures that observed signals genuinely represent ITPKC rather than non-specific binding or cross-reactivity with other proteins.

How does ITPKC function as a regulator of T-cell activation?

ITPKC plays a sophisticated role in regulating T-cell activation through the Ca²⁺/NFAT signaling pathway. Research has established that ITPKC functions as a negative regulator in this process . The mechanism involves the phosphorylation of inositol 1,4,5-trisphosphate (IP3), which is a crucial second messenger in T-cell receptor (TCR) signaling . When the TCR complex is stimulated, IP3 is generated and increases intracellular Ca²⁺ through interaction with IP3 receptors on the endoplasmic reticulum . This Ca²⁺ influx leads to nuclear translocation of NFAT, activating transcription of interleukin-2 and other cytokines essential for immune response .

By phosphorylating IP3, ITPKC effectively reduces the amount of this messenger available to trigger calcium release, thereby attenuating T-cell activation. This regulatory function makes ITPKC a potential target for studying immune dysregulation in various pathological conditions . When designing experiments to investigate this regulatory function, researchers should consider:

• Monitoring ITPKC expression before and after T-cell stimulation using properly validated antibodies

• Measuring calcium flux in cells with normal versus altered ITPKC expression/function

• Assessing downstream activation markers such as NFAT nuclear translocation and cytokine production

What is the significance of ITPKC polymorphisms in disease pathogenesis?

Genetic studies have identified a functional single nucleotide polymorphism (SNP) in the ITPKC gene (rs28493229, also referred to as itpkc_3) that is significantly associated with Kawasaki disease susceptibility and an increased risk of coronary artery lesions in both Japanese and US children . Transfection experiments demonstrated that the C allele of itpkc_3 reduces splicing efficiency of the ITPKC mRNA . This reduced efficiency may contribute to immune hyper-reactivity in Kawasaki disease by impairing the negative regulatory function of ITPKC in T-cell activation .

These findings highlight the importance of considering genetic variations when studying ITPKC function in disease contexts. Researchers investigating ITPKC in disease pathogenesis should incorporate genotyping of relevant polymorphisms and correlate these with functional outcomes measured using ITPKC antibodies.

What methodological approaches can optimize ITPKC antibody performance in Western blotting?

Western blotting with ITPKC antibodies presents several technical challenges that researchers should address through careful methodology:

• Molecular weight considerations: The calculated molecular weight of ITPKC is 75 kDa, but the observed molecular weight in Western blots is typically 100-105 kDa . This discrepancy should be taken into account when interpreting bands.

• Optimized sample preparation:

  • Use fresh tissue or cells whenever possible

  • Include appropriate protease and phosphatase inhibitors in lysis buffers

  • Ensure complete protein denaturation with proper heating in sample buffer

• Dilution optimization: Start with the recommended dilution range (1:500-1:2000) and perform a dilution series to determine optimal concentration for your specific sample type.

• Blocking strategy: Use 5% non-fat dry milk or BSA in TBST, depending on the specific antibody recommendations.

• Enhanced detection methods: For low-abundance detection, consider:

  • Extended exposure times with chemiluminescent substrates

  • Signal amplification systems

  • More sensitive detection reagents for weakly expressed ITPKC

• Controls and validation:

  • Include positive controls such as MDA-MB-453s cells or human heart tissue lysates

  • Run negative controls and blocking peptide controls where appropriate

  • Verify band specificity through parallel detection with another ITPKC antibody targeting a different epitope

How can ITPKC antibodies be utilized to investigate its role in Kawasaki disease pathogenesis?

ITPKC has emerged as a significant factor in Kawasaki disease (KD) pathogenesis, particularly through its role in T-cell activation regulation. Researchers can utilize ITPKC antibodies in multiple approaches to investigate this connection:

• Immunohistochemical analysis:

  • Compare ITPKC expression patterns in cardiovascular tissues from KD patients versus controls

  • Co-stain with T-cell markers to assess localization in inflammatory infiltrates

  • Use dilutions of 1:50-1:500 with appropriate antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

• Genetic-protein correlation studies:

  • Stratify samples based on known ITPKC polymorphisms, particularly rs28493229

  • Quantify ITPKC protein expression levels in relation to genotype

  • Assess splicing efficiency in patient-derived samples with different genotypes

• Functional studies in patient-derived cells:

  • Isolate PBMCs from KD patients and controls

  • Compare ITPKC expression levels before and after stimulation with PMA/ionomycin

  • Correlate calcium flux responses with ITPKC expression/polymorphism status

  • Measure downstream activation markers of the Ca²⁺/NFAT pathway

• Animal models:

  • Utilize ITPKC antibodies in murine models of Kawasaki disease

  • Assess cardiovascular lesion development in relation to ITPKC expression

  • Test therapeutic interventions targeting the ITPKC pathway

This multi-faceted approach allows researchers to connect genetic susceptibility factors with functional outcomes and potentially identify new therapeutic targets for KD treatment.

What troubleshooting strategies address common challenges with ITPKC antibodies?

Researchers may encounter several challenges when working with ITPKC antibodies. The following troubleshooting strategies address common issues:

For immunoprecipitation applications specifically, researchers should use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate and consider pre-clearing lysates to reduce non-specific binding. For immunofluorescence, a more concentrated antibody dilution (1:20-1:200) is typically required compared to Western blotting .