ITPR1 Antibody, HRP conjugated

What is ITPR1 and what cellular functions does it regulate?

ITPR1 (inositol 1,4,5-trisphosphate receptor type 1) is an intracellular receptor channel that mediates calcium release from the endoplasmic reticulum following stimulation by inositol 1,4,5-trisphosphate. The canonical human protein has a length of 2758 amino acid residues and a molecular mass of approximately 313.9 kDa . ITPR1 is primarily localized in the endoplasmic reticulum membrane, cytoplasmic vesicles, and cytoplasm . It plays a crucial role in calcium signaling pathways that regulate numerous cellular processes including neuronal function, gene expression, and cellular metabolism. Alternative splicing produces at least 8 different isoforms of this protein, allowing for tissue-specific functions . The protein undergoes various post-translational modifications including glycosylation, ubiquitination, palmitoylation, and phosphorylation, which can significantly alter its function and regulation .

What is the difference between unconjugated and HRP-conjugated ITPR1 antibodies?

Unconjugated ITPR1 antibodies contain no attached reporter molecules and require secondary detection systems in applications such as Western blotting, immunohistochemistry, and immunofluorescence. In contrast, HRP (horseradish peroxidase) conjugated ITPR1 antibodies have the enzyme directly attached to the antibody molecule, eliminating the need for secondary antibodies in detection systems .

The primary advantages of HRP-conjugated antibodies include:

• Simplified workflow with fewer incubation and washing steps

• Reduced background signal by eliminating potential cross-reactivity from secondary antibodies

• Enhanced sensitivity in enzyme-based detection systems such as ELISA

• Greater specificity in complex tissue samples

The ITPR1 antibody with HRP conjugation is specifically recommended for ELISA applications according to product information, while the unconjugated version is typically used for a broader range of applications including Western blot, immunoprecipitation, and immunohistochemistry .

What species reactivity can be expected when using ITPR1 antibodies?

Commercial ITPR1 antibodies demonstrate verified reactivity with human, mouse, and rat samples across multiple sources . According to citation data, researchers have also successfully used these antibodies with samples from chicken, sheep, and duck models . When selecting an ITPR1 antibody for cross-species applications, researchers should note that:

Sequence homology analysis between species can provide additional information about potential cross-reactivity, but experimental validation is always recommended when working with species not explicitly listed in the product specifications .

What are the recommended dilutions and experimental conditions for ITPR1 antibody, HRP conjugated in various applications?

The optimal dilution of ITPR1 antibody, HRP conjugated, varies by application and manufacturer specifications. Based on available data, the following guidelines can help researchers optimize their experimental protocols:

For ELISA applications:

• The HRP-conjugated ITPR1 antibody is specifically recommended for ELISA applications

• While specific dilutions for the HRP-conjugated version aren't provided in the search results, typical starting ranges for similar antibodies would be 1:1000-1:5000

For comparative purposes, the recommended dilutions for unconjugated ITPR1 antibodies in various applications are:

These dilutions should serve as starting points, and researchers should perform optimization experiments to determine the ideal concentration for their specific samples and detection systems .

How should ITPR1 antibody, HRP conjugated be stored to maintain optimal activity?

Proper storage of ITPR1 antibody, HRP conjugated, is crucial for maintaining its specificity and activity. According to manufacturer recommendations:

• Store the antibody at -20°C for long-term preservation

• The antibody remains stable for approximately 12 months after shipment when stored properly

• For the unconjugated version, aliquoting is deemed unnecessary for -20°C storage according to some manufacturers, but this may not apply to the HRP-conjugated version which might be more sensitive to freeze-thaw cycles

• The antibody is typically supplied in PBS buffer with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability

• Avoid repeated freeze-thaw cycles which can damage both the antibody and the HRP enzyme conjugate

• Some smaller quantity formats (20μl) may contain 0.1% BSA as a stabilizer

For working solutions, store at 4°C and use within 1-2 weeks. Always centrifuge briefly before opening the vial to ensure the solution is at the bottom of the tube .

What positive and negative controls should be included when using ITPR1 antibody, HRP conjugated?

Proper experimental controls are essential for validating results obtained with ITPR1 antibody, HRP conjugated:

Positive Tissue/Cell Controls:

• Mouse brain tissue: Consistently shows positive detection in Western blot and immunoprecipitation

• Mouse liver tissue: Verified positive in Western blot applications

• Human brain tissue: Positive in immunohistochemistry applications

• Human testis tissue: Reported positive in immunohistochemistry

• HepG2 cells: Successful detection in flow cytometry (intracellular)

Negative Controls:

• Primary Antibody Omission: Replace primary antibody with buffer or isotype-matched immunoglobulin

• Blocking Peptide Control: Pre-incubate the antibody with its specific immunizing peptide

• ITPR1 Knockout/Knockdown Models: Use ITPR1-deficient samples where available

• Non-expressing Tissues: Include tissues known to express minimal ITPR1

Additional Verification Controls:

• Perform parallel experiments using another validated anti-ITPR1 antibody targeting a different epitope

• Include both positive and negative samples in multiplexed detection systems

• For HRP-conjugated antibodies specifically, include an enzyme activity control to ensure the HRP component is functional

Why might the observed molecular weight of ITPR1 differ from the calculated theoretical weight?

The calculated molecular weight of ITPR1 is approximately 313.9-314 kDa, but observed molecular weights in experimental settings typically range from 290-315 kDa . This discrepancy can be attributed to several factors:

• Post-translational modifications: ITPR1 undergoes multiple post-translational modifications including glycosylation, ubiquitination, palmitoylation, and phosphorylation that can significantly affect migration patterns in gel electrophoresis

• Alternative splicing: The presence of 8 different isoforms due to alternative splicing can result in proteins of different molecular weights

• Protein conformation: The three-dimensional structure of proteins can affect their migration in SDS-PAGE

• Experimental conditions: Variations in gel percentage, buffer systems, and electrophoresis conditions can influence protein migration

As noted by one manufacturer: "The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size... If a protein in a sample has different modified forms at the same time, multiple bands may be detected on the membrane."

When working with ITPR1, researchers should anticipate potential band shifts and validate specific bands using positive controls from tissues known to express ITPR1, such as brain and liver tissues .

How can researchers differentiate between ITPR1 isoforms using antibodies?

ITPR1 exists in multiple isoforms due to alternative splicing, with at least 8 different variants reported . Differentiating between these isoforms requires careful consideration of:

• Epitope specificity: Determine which region of ITPR1 the antibody recognizes and whether this region is conserved across isoforms or specific to certain variants

• Molecular weight discrimination: Different isoforms may exhibit distinct molecular weights that can be resolved on Western blots using appropriate gel concentration and running conditions

• Isoform-specific antibodies: When available, use antibodies specifically raised against unique sequences in particular isoforms

• Combined approaches: Employ a combination of techniques including:

  • RT-PCR with isoform-specific primers to correlate protein detection with mRNA expression

  • Immunoprecipitation followed by mass spectrometry to confirm isoform identity

  • Tissue-specific analysis based on known differential expression patterns of isoforms

• Knockout validation: Use tissues or cells with specific isoforms knocked out as negative controls

Since standard commercial antibodies like those described in the search results may not readily distinguish between isoforms, researchers studying isoform-specific functions should consider custom antibodies raised against unique regions or supplementary techniques for definitive isoform identification .

What are the considerations when studying ITPR1 in neural tissues with HRP-conjugated antibodies?

ITPR1 is prominently expressed in neural tissues, particularly in Purkinje cells of the cerebellum, making it an important target in neuroscience research . When using HRP-conjugated ITPR1 antibodies in neural tissue studies, consider the following specialized approaches:

• Tissue preparation: For optimal detection in neural tissues:

  • Use antigen retrieval with TE buffer pH 9.0 for fixed tissues

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

• Signal localization: ITPR1 distribution in neural cells is distinctive:

  • Abundant in Purkinje cell somata, dendrites, and axons

  • Present in the molecular layer, Purkinje cell layer, and white matter of cerebellum

• Background considerations:

  • Neural tissues often have high endogenous peroxidase activity requiring effective quenching

  • Pre-incubation with hydrogen peroxide solution (0.3-3%) is recommended before antibody application

  • Multiple PBS washes help reduce nonspecific background

• Detection methods:

  • DAB (3,3'-diaminobenzidine) substrate is commonly used with HRP-conjugated antibodies

  • For co-localization studies in neural tissues, consider spectral separation strategies when using multiple antibodies

• Disease-relevant considerations:

  • ITPR1 autoantibodies have been implicated in autoimmune cerebellitis

  • Mutations in ITPR1 are associated with spinocerebellar ataxia

When studying these disease-relevant contexts, carefully validate antibody specificity to distinguish between endogenous ITPR1 and potential autoantibodies present in patient samples .

How is ITPR1 implicated in neurological disorders and what research methods best capture these relationships?

ITPR1 dysfunction has been implicated in several neurological disorders, with particularly strong associations to cerebellar ataxias. Research approaches using ITPR1 antibodies can provide valuable insights into these disease mechanisms:

• Spinocerebellar ataxias: Mutations in ITPR1 have been directly linked to spinocerebellar ataxia types 15, 16, and 29, with and without cognitive decline . Research applications include:

  • Immunohistochemistry of patient tissues to assess ITPR1 expression levels and localization

  • Western blot analysis to detect truncated or abnormal forms of the protein

  • Co-immunoprecipitation to investigate disrupted protein-protein interactions

• Autoimmune cerebellitis: ITPR1 can be the target of autoantibodies that disrupt cerebellar function . Detection methods include:

  • Indirect immunofluorescence using patient sera on rodent cerebellar sections

  • ELISA with recombinant ITPR1 protein to quantify autoantibody levels

  • Cell-based assays with HEK293 cells expressing ITPR1

• Calcium signaling disorders: As ITPR1 mediates calcium release from the endoplasmic reticulum, its dysfunction affects calcium homeostasis . Research approaches include:

  • Calcium imaging in cells with normal vs. altered ITPR1 expression

  • Correlation of ITPR1 antibody staining patterns with abnormal calcium signaling

  • Pharmacological manipulation of ITPR1 in disease models

For studying these conditions, researchers should consider using multiple detection methods, as each provides complementary information about ITPR1's role in disease pathogenesis .

How can ITPR1 antibodies be used to study calcium dysregulation in neurodegenerative diseases?

ITPR1 plays a critical role in calcium homeostasis and signaling, making ITPR1 antibodies valuable tools for investigating calcium dysregulation in neurodegenerative contexts:

• Co-localization studies: Using ITPR1 antibodies in combination with markers for:

  • ER stress (e.g., CHOP, BiP/GRP78)

  • Calcium binding proteins (e.g., calbindin, calretinin)

  • Apoptotic markers (e.g., cleaved caspase-3)

  • These approaches help establish relationships between ITPR1-mediated calcium release and cell death pathways

• Quantitative analysis in disease progression:

  • Compare ITPR1 expression levels and distribution at different disease stages

  • Correlate changes in ITPR1 localization with calcium imaging data

  • Analyze ITPR1 post-translational modifications in disease states

• Methodological considerations for calcium dysregulation studies:

  • For immunohistochemistry: Use ITPR1 antibody dilutions of 1:50-1:500

  • For Western blot analysis: 1:200-1:1000 dilution to track expression changes

  • For flow cytometry: 0.20 μg per 10^6 cells to quantify cellular ITPR1 levels

• Therapeutic intervention models:

  • ITPR1 antibodies can help assess whether experimental treatments normalize:

    • ITPR1 expression levels

    • Subcellular localization

    • Association with regulatory proteins

    • Calcium signaling patterns

By combining ITPR1 antibody-based detection with functional calcium measurements, researchers can develop a more comprehensive understanding of how alterations in this receptor contribute to neurodegenerative disease progression .

What is the significance of ITPR1 autoantibodies in neurological autoimmune conditions?

Research has identified that autoantibodies against ITPR1 can be associated with autoimmune cerebellar ataxia, representing an important diagnostic and research target:

• Clinical significance:

  • High-titer (up to 1:5,000) IgG antibodies, mainly IgG1 subclass, against ITPR1 have been detected in patients with cerebellar ataxia

  • These autoantibodies bind to Purkinje cell somata, dendrites, and axons

  • The binding pattern affects the molecular layer, Purkinje cell layer, and white matter of the cerebellum

• Detection methodologies:

  • Immunohistochemistry on mouse, rat, porcine, and monkey cerebellum sections

  • Specific neutralization experiments using preadsorption with purified ITPR1

  • Dot-blot assays with purified ITPR1 protein

  • Recombinant cell-based immunofluorescence assays using HEK293 cells expressing ITPR1

• Differential diagnosis considerations:

  • ITPR1 autoantibodies produce a binding pattern similar to anti-ARHGAP26 autoantibodies

  • Confirmation requires specific testing to distinguish between these entities

  • Control studies should rule out other paraneoplastic and non-paraneoplastic anti-neural autoantibodies

• Research methodologies for autoantibody studies:

  • For tissue-based assays: Apply patient serum (diluted) to brain sections for 1 hour

  • For antibody detection: Use fluorescein isothiocyanate (FITC), Alexa Fluor® 488, or Alexa Fluor® 568-conjugated secondary antibodies

  • For double staining: Combine commercial anti-ITPR1 antibodies with patient sera to confirm target identity

These findings suggest that autoimmunity against ITPR1 plays a role in the pathogenesis of autoimmune cerebellitis and provides a valuable diagnostic marker for researchers and clinicians investigating cerebellar ataxias of unknown etiology .

How can researchers optimize ITPR1 antibody performance in multiplex detection systems?

Multiplex detection systems allow simultaneous analysis of multiple targets, providing valuable contextual information about ITPR1 in relation to other proteins. To optimize ITPR1 antibody performance in these systems:

• Antibody selection criteria:

  • Choose antibodies raised in different host species to avoid cross-reactivity

  • For HRP-conjugated ITPR1 antibody, pair with antibodies using different detection systems (e.g., fluorescence)

  • Verify that epitopes are accessible under the same fixation and permeabilization conditions

• Detection optimization:

  • Carefully titrate each antibody independently before combining

  • For HRP-conjugated antibodies in chromogenic multiplexing, use substrates with distinct colors

  • Consider tyramide signal amplification (TSA) for enhanced sensitivity while maintaining multiplexing capability

• Buffer and protocol harmonization:

  • Use common buffers compatible with all antibodies in the panel

  • Optimize antigen retrieval protocols that work for all targets

  • Sequential incubation may be necessary if antibodies require different conditions

• Validation approaches:

  • Always include single-stained controls alongside multiplex specimens

  • Use spectral imaging and unmixing for fluorescent applications

  • Confirm staining patterns match those seen in single-antibody experiments

• Specific considerations for ITPR1:

  • Given its large molecular weight (313.9 kDa), ensure complete protein transfer in Western blot multiplexing

  • For tissue multiplexing, note that ITPR1 shows specific localization patterns in neural tissues that should be preserved

These strategies help ensure reliable multiplex detection while maintaining the specificity and sensitivity of ITPR1 antibody staining.

What are the best approaches for confirming ITPR1 antibody specificity in experimental systems?

Confirming antibody specificity is crucial for generating reliable research data. For ITPR1 antibodies, including HRP-conjugated versions, employ these validation strategies:

• Genetic validation approaches:

  • Test antibody in ITPR1 knockout/knockdown models

  • Use CRISPR-Cas9 edited cell lines with ITPR1 deletion

  • Compare staining in tissues with known differential expression patterns

• Biochemical validation methods:

  • Preabsorption studies with immunizing peptide or purified protein

  • Western blot analysis confirming bands at expected molecular weight (290-315 kDa)

  • Immunoprecipitation followed by mass spectrometry identification

• Orthogonal technique confirmation:

  • Correlate protein detection with mRNA expression (qPCR, RNA-seq)

  • Compare localization patterns using different anti-ITPR1 antibodies targeting distinct epitopes

  • Use alternative detection methods (e.g., in situ hybridization) to confirm expression patterns

• Application-specific controls:

  • For ELISA: Include standard curves with recombinant ITPR1

  • For IHC/IF: Test across multiple tissue types with known ITPR1 expression profiles

  • For WB: Include positive control lysates from brain or liver tissue

• Documentation requirements:

  • Record detailed antibody information including catalog number, lot, dilution

  • Document all validation experiments performed

  • Report both positive and negative findings to build a complete specificity profile

Comprehensive validation using multiple approaches provides the strongest evidence for antibody specificity and ensures reliable experimental results .

How can post-translational modifications of ITPR1 affect antibody recognition and experimental interpretation?

ITPR1 undergoes several post-translational modifications (PTMs) that can significantly impact antibody recognition and experimental outcomes:

• Types of ITPR1 post-translational modifications:

  • Glycosylation: Affects protein folding and trafficking

  • Ubiquitination: Influences protein degradation

  • Palmitoylation: Alters membrane association

  • Phosphorylation: Regulates channel activity and protein interactions

• Impact on antibody recognition:

  • Epitope masking: PTMs can physically block antibody access to recognition sites

  • Conformational changes: Modifications may alter protein structure, affecting conformational epitopes

  • Modified epitopes: If an antibody's target sequence contains a modification site, recognition may be impaired or enhanced

• Experimental considerations:

  • Verify antibody epitope location relative to known modification sites

  • Test antibody performance under conditions that preserve or remove specific modifications

  • Consider using phosphatase or glycosidase treatments to assess modification-dependent recognition

• Interpretation strategies:

  • When observing unexpected band patterns, consider PTM heterogeneity as a possible explanation

  • Multiple bands between 290-315 kDa may represent differently modified ITPR1 forms

  • Tissue-specific differences in band patterns may reflect differential PTM processing

• Advanced approaches for PTM-aware analysis:

  • Use modification-specific antibodies in parallel with total ITPR1 antibodies

  • Employ 2D gel electrophoresis to separate PTM variants

  • Consider mass spectrometry to characterize specific modifications in immunoprecipitated ITPR1

Understanding how PTMs affect ITPR1 antibody recognition is essential for accurate data interpretation, particularly in studies comparing ITPR1 across different physiological or pathological states .

What emerging research areas will benefit from advanced ITPR1 antibody applications?

As our understanding of calcium signaling pathways and ITPR1 biology continues to evolve, several research frontiers can benefit from advanced ITPR1 antibody applications:

• Neurodegenerative disease mechanisms:

  • Investigating ITPR1's role in protein misfolding disorders

  • Studying calcium dyshomeostasis in Alzheimer's, Parkinson's, and ALS

  • Exploring the therapeutic potential of ITPR1 modulation

• Autoimmune disorder diagnostics:

  • Developing standardized assays for detecting anti-ITPR1 autoantibodies

  • Creating diagnostic algorithms for cerebellar ataxias of unknown etiology

  • Establishing correlations between autoantibody titers and clinical outcomes

• Calcium signaling microdomains:

  • Mapping ITPR1 distribution relative to other calcium channels using super-resolution microscopy

  • Characterizing ITPR1 interactome in different cellular compartments

  • Studying dynamic changes in ITPR1 localization under physiological stimuli

• Personalized medicine approaches:

  • Correlating ITPR1 expression patterns with treatment responses

  • Developing companion diagnostics for calcium-targeting therapeutics

  • Identifying patient subgroups based on ITPR1 variants or modifications

• Methodological advances:

  • Integration with emerging technologies:

    • Developing proximity labeling approaches for ITPR1 interaction networks

    • Creating live-cell imaging tools using anti-ITPR1 antibody fragments

    • Establishing high-throughput screening platforms for ITPR1 modulators