ITPRIPL2 Antibody

What is ITPRIPL2 and why is it important in biological research?

ITPRIPL2 is a single-pass type I membrane protein that interacts with inositol 1,4,5-trisphosphate receptors (IP3Rs), which are crucial calcium channels in the endoplasmic reticulum. Research indicates that ITPRIPL2 plays a significant role in calcium signaling and inflammatory responses. The protein has gained research interest due to its potential involvement in neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS), where genetic ablation of IP3R2 has been shown to increase cytokines and exacerbate disease progression . Understanding ITPRIPL2's function provides insights into calcium homeostasis and inflammatory pathways, making it a valuable target for both basic and translational research.

Which applications are most suitable for ITPRIPL2 antibodies?

ITPRIPL2 antibodies have been validated primarily for:

For optimal results, researchers should verify antibody performance in their specific model systems as reactivity has been confirmed in human and mouse samples .

How should ITPRIPL2 antibodies be stored and handled for maximum stability?

ITPRIPL2 antibodies are typically supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide . For optimal stability and performance:

• Store antibodies at -20°C for up to one year from receipt date

• Avoid repeated freeze-thaw cycles which can diminish antibody activity

• For short-term storage (1-2 weeks), antibodies can be kept at 4°C

• When handling, keep antibodies on ice and return to storage promptly

• If dividing into aliquots, use sterile conditions to prevent contamination

Following these storage guidelines ensures maintained reactivity and specificity for the duration of experimental studies.

What controls should be included when validating ITPRIPL2 antibody specificity?

Robust validation of ITPRIPL2 antibodies requires multiple controls:

• Positive tissue controls: Human tissues with known ITPRIPL2 expression (based on Human Protein Atlas data)

• Negative controls: Omission of primary antibody or use of isotype-matched IgG

• Knockdown/knockout validation: Comparison of signal between wildtype and ITPRIPL2-depleted samples

• Peptide competition: Pre-incubation of antibody with immunizing peptide should abolish specific signal

• Cross-reactivity assessment: Testing on protein arrays (like the 364 human recombinant protein fragments array used for Prestige Antibodies)

The Human Protein Atlas project provides extensive validation data for some commercial ITPRIPL2 antibodies, including immunohistochemistry results from 44 normal human tissues and 20 common cancer types .

How can researchers optimize Western blot protocols for ITPRIPL2 detection?

For optimal Western blot detection of ITPRIPL2:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylation states

  • Denature samples at 95°C for 5 minutes in reducing conditions

• Gel selection and transfer:

  • Use 10-12% SDS-PAGE gels

  • Transfer to PVDF membranes (preferred over nitrocellulose for this protein)

  • Perform wet transfer at 100V for 60-90 minutes

• Antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour

  • Dilute primary antibody to 0.04-0.4 μg/mL (approximately 1:500-1:2000)

  • Incubate overnight at 4°C with gentle rocking

  • Use appropriate HRP-conjugated secondary antibody (typically 1:5000-1:10000)

• Detection optimization:

  • For weak signals, extend primary antibody incubation time

  • Consider using enhanced chemiluminescence (ECL) substrates designed for low-abundance proteins

  • If background is high, increase washing steps or add 0.05% Tween-20 to antibody dilution buffer

These optimization steps should result in specific detection of ITPRIPL2 at the expected molecular weight.

How has ITPRIPL2 been implicated in ALS pathology, and what experimental approaches can investigate this relationship?

Research indicates that ITPRIPL2's gene product, IP3R2, plays a significant role in ALS pathology:

• Clinical evidence: Increased ITPRIPL2 gene expression has been detected in blood samples of sporadic ALS patients .

• Animal model findings:

  • ITPRIPL2 expression is upregulated in ventral spinal cords of symptomatic and end-stage SOD1^G93A mice

  • Genetic ablation of IP3R2 in SOD1^G93A mice results in:

    • Shorter disease duration and decreased survival (dose-dependent effect)

    • Increased innate immunity markers

    • Elevated pro-inflammatory cytokines (IFNγ and IL1α)

• Experimental approaches to investigate ITPRIPL2 in ALS:

  • Gene expression analysis: qPCR to quantify ITPRIPL2 mRNA levels in spinal cord tissues

  • Protein localization: IHC/IF using ITPRIPL2 antibodies on spinal cord sections

  • Calcium signaling assays: Measure IP3-induced calcium release in presence/absence of ITPRIPL2

  • Cytokine profiling: Quantify inflammatory markers (IFNγ, IL6, IL1α) in serum and CNS

  • Flow cytometry: Assess immune cell populations, particularly focusing on Ly6C^hi and Ly6C^lo monocytes

These approaches can help elucidate the protective role of ITPRIPL2 upregulation as a compensatory mechanism during neuroinflammation.

What methodological considerations are important when studying ITPRIPL2 in neuroinflammatory conditions?

When investigating ITPRIPL2 in neuroinflammatory settings, researchers should consider:

• Model selection:

  • Acute models: Photothrombotic cortical stroke (showed increased IP3R2 expression in penumbra)

  • Chronic models: EAE (experimental autoimmune encephalomyelitis), SOD1^G93A mice

  • In vitro models: LPS-stimulated astrocytes or macrophages

• Specificity controls:

  • Monitor expression of all IP3R isoforms (IP3R1, IP3R2, IP3R3) as ITPRIPL2 interacts specifically with IP3R2

  • Verify antibody cross-reactivity between IP3R isoforms

• Timing considerations:

  • In neurodegeneration models, assess multiple disease stages (pre-symptomatic, early symptomatic, end-stage)

  • For acute inflammation, establish appropriate time course (e.g., 6h, 12h, 24h, 48h post-stimulus)

• Cell type-specific analysis:

  • Distinguish between neuronal and glial ITPRIPL2 expression

  • Consider cell isolation techniques (FACS, magnetic separation) before molecular analysis

• Functional readouts:

  • Calcium imaging with IP3-generating agonists

  • Cytokine production measurement (ELISA, multiplex assays)

  • Assessment of inflammatory cell infiltration and activation markers

These methodological considerations ensure robust and reproducible data when studying ITPRIPL2's role in neuroinflammatory conditions.

How can antibodyomics approaches incorporating ITPRIPL2 antibodies contribute to understanding immune responses?

Recent advances in antibodyomics provide powerful tools for studying complex immune responses, applicable to ITPRIPL2 research:

• High-throughput immunoglobulin heavy chain (IgH) repertoire sequencing:

  • Enables tracking of specific antibody lineages targeting particular epitopes

  • Can identify shared antibody clonotypes across patient cohorts

  • Allows longitudinal studies of antibody persistence and evolution

• ITPRIPL2 antibody application in antibodyomics:

  • ITPRIPL2 antibodies can be used as control antibodies in establishing antibodyomics pipelines

  • As membrane proteins are common targets in autoimmune responses, ITPRIPL2 antibodyomics could reveal patterns in neuroinflammatory diseases

  • Combined with structural analysis, this approach can map immunodominant epitopes on membrane proteins

• Methodological workflow:

  • Isolate B cells from patient samples (blood, CSF, or affected tissues)

  • Perform IgH repertoire sequencing (>10 million sequences per sample)

  • Cluster sequences with known antibody specificities

  • Map epitopes using structural and bioinformatics analysis

  • Track antibody lineages over time or across patient populations

This approach has been successfully used to create comprehensive atlases of spike-targeting antibody lineages in COVID-19 research and could be adapted to study ITPRIPL2-targeting antibodies in autoimmune or inflammatory neurological conditions.

What are the technical challenges in studying ITPRIPL2's interaction with IP3R2, and how can antibodies help overcome these limitations?

Studying protein-protein interactions between ITPRIPL2 and IP3R2 presents several technical challenges:

• Structural complexity:

  • IP3R2 is a large tetrameric calcium channel (>2500 amino acids per monomer)

  • ITPRIPL2 is a membrane protein, making structural studies challenging

• Technical limitations and antibody-based solutions:

• Advanced methodological approaches:

  • Co-immunoprecipitation: Use anti-ITPRIPL2 antibodies to pull down protein complexes and detect IP3R2 association

  • FRET/BRET assays: Combine with antibody-based detection to verify interaction sites

  • Super-resolution microscopy: Use fluorescently-labeled antibodies to visualize co-localization at nanoscale resolution

  • Cryo-EM: Use antibody fragments (Fabs) to stabilize protein complexes for structural determination

These antibody-based approaches can overcome many of the inherent challenges in studying membrane protein interactions.

How can researchers address common issues with ITPRIPL2 antibody specificity and cross-reactivity?

When encountering specificity issues with ITPRIPL2 antibodies:

• Common specificity problems and solutions:

• Validation strategies:

  • Compare results using antibodies from different vendors targeting different epitopes

  • Perform peptide competition assays using the immunogen sequence

  • Test specificity against recombinant protein fragments

  • Include cross-reactivity controls for related proteins, especially other IP3R-interacting proteins

• Application-specific considerations:

  • For IHC: Test different antigen retrieval methods

  • For WB: Compare reducing vs. non-reducing conditions

  • For IF: Optimize fixation protocols (PFA vs. methanol)

Thorough validation using these approaches ensures reliable and reproducible results when studying ITPRIPL2.

How should conflicting data on ITPRIPL2 expression or function be interpreted in the context of different experimental models?

When faced with conflicting data on ITPRIPL2:

• Sources of experimental variability:

  • Different antibody epitopes may detect specific isoforms or post-translationally modified forms

  • Cell/tissue-specific expression patterns (e.g., ITPRIPL2 upregulation appears specific to certain cell types under inflammatory conditions)

  • Species differences in ITPRIPL2 sequence and function (human vs. mouse ortholog identity is 92%, but only 24% for rat)

• Systematic approach to resolving conflicts:

  • Experimental model comparison: Evaluate differences between in vitro cell lines, primary cultures, and in vivo models

  • Methodological assessment: Compare protein detection methods (WB/IHC/IF) with transcript analysis (qPCR/RNA-seq)

  • Temporal considerations: Assess whether discrepancies relate to different time points or disease stages

  • Genetic background effects: Consider strain/background differences in animal models

• Case study from literature: IP3R2 expression in ALS models

  • Evidence shows that IP3R2 is specifically upregulated in ALS spinal cord, while IP3R1 and IP3R3 show no changes

  • Genetic ablation of IP3R2 worsens disease in SOD1^G93A mice

  • This seemingly contradicts the protective effect of lowering calcium signaling

  • Resolution: IP3R2 upregulation is likely a compensatory protective mechanism against inflammation

• Recommended validation workflow:

  • Start with gene expression analysis (qPCR/RNA-seq)

  • Validate protein expression with multiple antibodies targeting different epitopes

  • Correlate with functional readouts (calcium signaling, inflammatory markers)

  • Consider genetic manipulation approaches (knockdown/knockout/overexpression)

This systematic approach helps reconcile apparently conflicting data and builds a more comprehensive understanding of ITPRIPL2 biology.

What emerging techniques could enhance ITPRIPL2 research beyond traditional antibody applications?

Several cutting-edge approaches show promise for advancing ITPRIPL2 research:

• CRISPR-based technologies:

  • CRISPR-Cas9 knock-in of fluorescent tags for live-cell imaging of endogenous ITPRIPL2

  • CRISPRi/CRISPRa for targeted modulation of ITPRIPL2 expression

  • Base editors for introducing specific point mutations to study structure-function relationships

• Advanced microscopy with antibody-based detection:

  • Lattice light-sheet microscopy for dynamic visualization of ITPRIPL2-IP3R interactions

  • Expansion microscopy combined with immunofluorescence for enhanced spatial resolution

  • Correlative light and electron microscopy (CLEM) using gold-conjugated antibodies

• Single-cell technologies:

  • CITE-seq (combining antibody detection with single-cell RNA-seq)

  • Mass cytometry (CyTOF) with metal-labeled ITPRIPL2 antibodies

  • Single-cell western blotting for heterogeneity analysis

• Biosensor development:

  • FRET-based biosensors incorporating ITPRIPL2-binding domains

  • Split fluorescent protein complementation assays to monitor interactions

  • Antibody-based proximity sensors for real-time interaction studies

These emerging techniques, when combined with traditional antibody applications, can provide unprecedented insights into ITPRIPL2 biology.

How might understanding ITPRIPL2 function contribute to therapeutic development for inflammatory and neurodegenerative diseases?

Based on current knowledge, ITPRIPL2 presents several promising therapeutic avenues:

• Mechanistic insights from IP3R2 research:

  • Genetic ablation of IP3R2 exacerbates ALS progression in mouse models

  • IP3R2 upregulation appears to be a protective mechanism against inflammation

  • IP3R2 modulates calcium signaling, which influences inflammatory cytokine production

• Potential therapeutic strategies:

  • Enhancing ITPRIPL2-IP3R2 interaction: Could reduce pro-inflammatory cytokine production

  • Modulating calcium signaling: Targeted approaches affecting specific IP3R isoforms

  • Cell-specific interventions: Targeting ITPRIPL2 function in specific immune cell populations (e.g., Ly6C^hi monocytes)

  • Combined approaches: Targeting multiple aspects of calcium-dependent inflammatory pathways

• Translational research approaches:

  • Screening for small molecules that enhance ITPRIPL2-IP3R2 interaction

  • Development of peptide mimetics based on ITPRIPL2 interaction domains

  • Targeted antibody-based therapeutic strategies (e.g., intrabodies)

  • Cell-based therapies exploiting ITPRIPL2's anti-inflammatory properties

• Biomarker potential:

  • ITPRIPL2 expression changes might serve as disease progression markers

  • Monitoring ITPRIPL2-specific antibody responses in autoimmune conditions

  • Combinatorial biomarker panels including ITPRIPL2 and related calcium signaling proteins

Understanding ITPRIPL2's protective role in inflammatory conditions could lead to novel therapeutic strategies focused on enhancing its function rather than inhibiting it, representing a paradigm shift in approach to inflammatory neurodegeneration.