ITPR1 Antibody, Biotin conjugated

What is ITPR1 and why is it significant in neurological research?

ITPR1 (inositol 1,4,5-triphosphate receptor, type 1) is a calcium channel receptor that mediates calcium release from the endoplasmic reticulum upon binding with inositol 1,4,5-trisphosphate. This receptor plays critical roles in numerous cellular signaling pathways that regulate neuronal function and development. The protein has a calculated molecular weight of 314 kDa, though it typically demonstrates an observed weight of 290-300 kDa in experimental conditions .

ITPR1 is abundantly expressed in cerebellar Purkinje cells but is also found in various other tissues including human brain tissue, testis tissue, mouse brain tissue, and liver tissue . Significantly, autoantibodies against ITPR1 have been identified in patients with autoimmune cerebellar ataxia, and mutations in ITPR1 have been implicated in spinocerebellar ataxia with and without cognitive decline . This makes ITPR1 detection particularly important for research into neurological disorders.

How does biotin conjugation enhance ITPR1 antibody functionality?

Biotin conjugation of ITPR1 antibodies creates a powerful research tool by exploiting the strong affinity between biotin and streptavidin/avidin. This conjugation enhances detection sensitivity through signal amplification, as multiple streptavidin molecules (conjugated to reporters like enzymes or fluorophores) can bind to each biotin molecule. For ITPR1 research, this is particularly valuable when:

• Detecting low-abundance ITPR1 in certain tissues or under specific conditions

• Performing multi-step labeling protocols where signal enhancement is needed

• Conducting ELISA assays, where biotin-conjugated ITPR1 antibodies show optimal performance

• Creating detection systems with flexible reporter options by changing only the streptavidin conjugate

The biotin-conjugated ITPR1 antibody (product code CSB-PA614538LD01HU) is specifically recommended for ELISA applications, where its binding characteristics provide reliable and sensitive detection of the target protein .

What are the optimal tissue preparation methods for ITPR1 detection using biotin-conjugated antibodies?

For optimal detection of ITPR1 using biotin-conjugated antibodies, tissue preparation varies by application:

For immunohistochemistry of brain tissue:

• Fixation: 4% paraformaldehyde is recommended for preserving ITPR1 epitopes

• Antigen retrieval: Use TE buffer at pH 9.0 as the primary method; alternatively, citrate buffer at pH 6.0 may be used

• Blocking: Employ 10% goat or donkey serum (depending on secondary antibodies) to reduce non-specific binding

• Section thickness: 5-10 μm sections typically provide optimal resolution for detecting ITPR1 in cerebellar tissues

For cell culture applications:

• Cells should be fixed with acetone for 10 minutes at room temperature

• For flow cytometry with intracellular staining, permeabilization with gentle detergents is essential

These protocols minimize autofluorescence and maximize signal-to-noise ratio when working with biotin-conjugated ITPR1 antibodies in neurological tissue samples.

How should I design controls when using ITPR1 antibody, biotin conjugated?

Proper experimental controls are critical when using biotin-conjugated ITPR1 antibodies:

Positive controls:

• Mouse brain tissue (particularly cerebellum) and liver tissue have been validated for ITPR1 detection

• Human brain and testis tissues have confirmed ITPR1 expression

• HepG2 cells are suitable for flow cytometry applications

Negative controls:

• Antibody omission control (all reagents except primary antibody)

• Isotype control (rabbit IgG at equivalent concentration)

• Blocking peptide control: Pre-adsorption of the antibody with purified ITPR1 protein before staining should eliminate specific signals

• Non-expressing tissue or knockout samples where available

Specificity validation:

• Dot-blot assay using purified ITPR1 protein with increasing dilutions (1:1.5, 1:3, 1:6, 1:12) to confirm binding specificity

• Western blot to verify the expected molecular weight (290-300 kDa)

• Comparison with alternative ITPR1 antibody clones to confirm staining pattern

What are the recommended dilution protocols for different applications?

While specific dilution guidelines for biotin-conjugated ITPR1 antibody (CSB-PA614538LD01HU) focus on ELISA applications , general dilution guidelines for ITPR1 antibodies that can inform biotin-conjugated applications include:

It is strongly recommended to titrate the biotin-conjugated antibody in each specific experimental system to obtain optimal results, as sensitivity can vary between tissue types and detection methods .

How can I optimize signal amplification when using biotin-conjugated ITPR1 antibodies?

Signal amplification with biotin-conjugated ITPR1 antibodies can be optimized through several advanced techniques:

• Tyramide Signal Amplification (TSA):

  • After primary biotin-conjugated ITPR1 antibody binding, use streptavidin-HRP

  • Apply tyramide solution to deposit additional biotin molecules at binding sites

  • Follow with a second round of streptavidin-reporter detection

  • This approach can increase sensitivity by 10-100 fold

• Multi-layer Streptavidin Systems:

  • Use alternating layers of biotin-labeled reagents and streptavidin

  • Each layer increases available binding sites for signal molecules

  • Particularly useful for detecting low ITPR1 expression in non-neuronal tissues

• Polymer-Based Detection:

  • Employ streptavidin-polymer conjugates carrying multiple reporter molecules

  • Reduces background while enhancing specific signal

  • Useful for colocalization studies with other calcium channel markers

• Optimization of Reaction Conditions:

  • Extend incubation times to 1-2 hours at room temperature or overnight at 4°C

  • Use gentle agitation to improve antibody penetration

  • Add protein carriers (0.1% BSA) to reduce non-specific binding

  • Increase washing steps (at least three washes in chilled PBS) between applications

These techniques should be validated with appropriate controls to ensure that amplification does not introduce artifacts.

What approaches can help resolve contradictory results when analyzing ITPR1 expression?

When encountering contradictory results in ITPR1 expression studies using biotin-conjugated antibodies, consider these methodological approaches:

• Antibody Validation:

  • Confirm specificity through pre-adsorption with purified ITPR1 (should eliminate signal)

  • Use dot-blot assays with purified ITPR1 at multiple dilutions to verify binding characteristics

  • Compare results with alternative antibodies targeting different ITPR1 epitopes

• Sample Preparation Assessment:

  • Evaluate effects of different fixation protocols on epitope accessibility

  • Compare fresh versus archived tissues

  • Consider antigen retrieval optimization with both TE buffer (pH 9.0) and citrate buffer (pH 6.0)

• Expression Analysis Through Multiple Methods:

  • Complement protein detection with mRNA analysis (RT-PCR, in situ hybridization)

  • Use recombinant cell-based assays with HEK293 cells expressing ITPR1

  • Implement mass spectrometry to confirm protein identity in immunoprecipitated samples

• Quantitative Standardization:

  • Develop calibration curves using purified ITPR1 protein

  • Implement digital image analysis with standardized intensity measurements

  • Use reference tissues with known ITPR1 expression levels

• Isoform-Specific Analysis:

  • Determine if contradictions stem from detection of different ITPR1 splice variants

  • Use computational analysis to predict epitope accessibility in different conformational states

  • Consider post-translational modifications that might affect antibody binding

What are the technical considerations for multiplex detection involving ITPR1?

Multiplex detection involving biotin-conjugated ITPR1 antibody requires careful technical planning:

• Preventing Cross-Reactivity:

  • When combining with other antibodies, choose those raised in different host species

  • If using multiple rabbit antibodies, sequential immunostaining with complete blocking between rounds is recommended

  • For neuronal studies, validated combinations include ITPR1 with calbindin-D, GFAP, and AQP4

• Fluorophore Selection:

  • When using streptavidin-fluorophore conjugates with biotin-ITPR1 antibody, select fluorophores with minimal spectral overlap

  • For ITPR1 detection in brain tissue, conjugates with excitation/emission away from tissue autofluorescence (avoid 350-450 nm range)

  • The CoraLite® Plus 488 conjugate (excitation/emission: 493 nm/522 nm) offers good separation from other common fluorophores

• Sequential Detection Protocols:

  • Apply biotin-conjugated ITPR1 antibody first when combining with non-biotinylated antibodies

  • Block all biotin binding sites before introducing other biotinylated reagents

  • Use streptavidin-conjugates last in the sequence to prevent cross-binding

• Imaging Considerations:

  • Employ spectral unmixing for closely overlapping signals

  • Use sequential scanning rather than simultaneous acquisition

  • Establish signal thresholds based on single-stained controls

A recommended multiplex panel for cerebellar studies would include biotin-conjugated ITPR1 antibody detected with streptavidin-far red, combined with direct-labeled antibodies for neuronal markers (e.g., calbindin-D) and glial markers (e.g., GFAP) .

How can I quantitatively assess ITPR1 expression levels using biotin-conjugated antibodies?

Quantitative assessment of ITPR1 expression using biotin-conjugated antibodies can be performed through several methodological approaches:

• ELISA-Based Quantification:

  • Develop a standard curve using recombinant ITPR1 protein at known concentrations

  • Biotin-conjugated ITPR1 antibody is particularly suited for sandwich ELISA systems

  • Implement four-parameter logistic regression for accurate concentration determination

  • Expected sensitivity can reach picogram levels with optimized protocols

• Image Analysis of Immunohistochemistry/Immunofluorescence:

  • Use digital image analysis software to measure:

    • Integrated optical density (IOD) of ITPR1 staining

    • Area percentage with positive staining

    • Mean fluorescence intensity in defined cellular compartments

  • Normalize to internal control proteins or housekeeping genes

  • Apply threshold consistency across all samples

• Flow Cytometry Quantification:

  • Use calibration beads with known quantities of fluorophore

  • Convert median fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF)

  • For intracellular ITPR1 detection, optimize with 0.20-0.40 μg antibody per 10^6 cells

  • Calculate relative expression compared to standard cell lines

• Western Blot Densitometry:

  • If using biotin-streptavidin detection systems in Western blots

  • Establish linear range of detection using purified ITPR1 dilution series

  • Normalize to loading controls (e.g., GAPDH, β-actin)

  • Account for the high molecular weight of ITPR1 (290-300 kDa) when optimizing transfer conditions

Each quantification method should include technical replicates and appropriate statistical analysis to ensure reproducibility.

What methods can differentiate between ITPR1 isoforms and post-translational modifications?

Differentiating between ITPR1 isoforms and post-translational modifications requires specialized analytical approaches:

• Isoform Discrimination:

  • High-resolution SDS-PAGE with extended separation time for the high molecular weight range (290-300 kDa)

  • Complementary RT-PCR analysis with isoform-specific primers

  • Mass spectrometry analysis of immunoprecipitated ITPR1 to identify specific peptide sequences

  • Recombinant expression systems with defined ITPR1 variants for comparison

• Phosphorylation Analysis:

  • Combined use of biotin-conjugated ITPR1 antibody with phospho-specific antibodies

  • Phosphatase treatment controls to confirm phosphorylation-dependent signals

  • Phos-tag™ SDS-PAGE to separate phosphorylated from non-phosphorylated forms

  • Correlation with functional calcium imaging to connect modification status with channel activity

• Other Post-Translational Modifications:

  • Glycosylation assessment through lectin binding assays or deglycosylation enzymes

  • Ubiquitination detection using co-immunoprecipitation with ubiquitin antibodies

  • S-nitrosylation analysis with biotin-switch technique

  • Correlation of modifications with cellular compartmentalization using subcellular fractionation

• Functional Correlations:

  • Patch-clamp electrophysiology to correlate modifications with channel conductance

  • Calcium imaging to assess channel functionality in relation to modifications

  • Protein-protein interaction studies to identify modification-dependent binding partners

These advanced analyses provide deeper insights into ITPR1 regulation beyond simple expression levels, which is particularly relevant for neurological disease research.

How does biotin-conjugated ITPR1 antibody compare with other conjugates for specific applications?

Comparative analysis of different ITPR1 antibody conjugates reveals application-specific advantages:

For neurological tissue samples, biotin-conjugated ITPR1 antibody offers superior sensitivity when detecting low-abundance targets, while HRP conjugates may be preferable for rapid screening. The choice should be guided by specific experimental requirements and available detection systems.

What methodological adaptations are needed when transitioning from animal to human tissue samples?

Transitioning ITPR1 detection methods from animal to human tissues requires specific methodological adaptations:

• Antigen Retrieval Optimization:

  • Human tissues typically require more stringent antigen retrieval

  • For ITPR1 detection in human brain tissue, TE buffer at pH 9.0 is recommended as the primary method

  • Alternatively, citrate buffer at pH 6.0 can be used with extended retrieval times

• Antibody Validation Strategies:

  • Confirm cross-reactivity with human ITPR1 (validated for biotin-conjugated antibodies)

  • Verify antibody specificity using human recombinant ITPR1 expressed in HEK293 cells

  • Compare staining patterns with established human ITPR1 expression profiles

• Background Reduction Techniques:

  • Human tissues often exhibit higher autofluorescence and endogenous biotin

  • Implement blocking of endogenous biotin/avidin using commercial blocking kits

  • Consider extended blocking (10% normal serum) to reduce non-specific binding

  • Use Sudan Black B or TrueBlack® to reduce autofluorescence in brain tissue

• Ethical and Methodological Considerations:

  • Account for post-mortem interval effects on epitope preservation

  • Consider fixation differences between clinical specimens and research samples

  • Address batch effects through standardized processing

  • Increase technical replicates when working with limited human samples

When transitioning to human cerebellum samples for ITPR1 detection, researchers should expect similar staining patterns as seen in animal models, with strong signals in the molecular layer, Purkinje cell layer, and white matter , though signal intensity may require optimization.

How can ITPR1 antibody data be integrated with functional calcium imaging studies?

Integration of biotin-conjugated ITPR1 antibody data with functional calcium imaging creates powerful research synergies:

• Sequential Analysis Protocols:

  • Perform live calcium imaging with indicators like Fluo-4 or GCaMP

  • Fix and process the same samples for ITPR1 immunodetection

  • Use registration algorithms to align functional and immunohistochemical datasets

  • Correlate ITPR1 expression levels with calcium response amplitudes and kinetics

• Combined Imaging Approaches:

  • Implement fixable calcium indicators compatible with subsequent immunostaining

  • Use spectral separation to distinguish calcium indicator signals from ITPR1 detection

  • Design experimental timelines that capture both acute calcium dynamics and long-term ITPR1 expression changes

• Analytical Integration Frameworks:

  • Develop computational models correlating ITPR1 density with calcium oscillation patterns

  • Implement machine learning algorithms to identify relationships between ITPR1 distribution and functional responses

  • Create visualization tools that overlay functional activity maps with ITPR1 expression maps

• Experimental Design Considerations:

  • Include pharmacological manipulations targeting ITPR1 (e.g., 2-APB, xestospongin C)

  • Compare wild-type with ITPR1 variant or knockdown models

  • Design longitudinal studies to track changes in both ITPR1 expression and calcium dynamics

For cerebellar studies, this integrated approach can reveal how ITPR1 expression patterns in Purkinje cells correlate with their calcium signaling properties, providing insights into how alterations in ITPR1 might contribute to cerebellar ataxia .