ORAI1 Antibody

What is ORAI1 and why are antibodies against it important for research?

ORAI1 functions as the pore-forming subunit of two major inward rectifying Ca²⁺ channels at the plasma membrane: Ca²⁺ release-activated Ca²⁺ (CRAC) channels and arachidonate-regulated Ca²⁺-selective (ARC) channels. It assembles with ORAI2 and ORAI3 to form hexameric CRAC channels that mediate Ca²⁺ influx upon depletion of endoplasmic reticulum Ca²⁺ stores, a process known as store-operated Ca²⁺ entry (SOCE) . ORAI1 mainly contributes to generating Ca²⁺ plateaus involved in sustained Ca²⁺ entry and is crucial for immune cell function, particularly in T-cells where it promotes immune responses by activating NFAT-dependent cytokine and chemokine transcription . ORAI1 antibodies are essential tools for studying these calcium signaling pathways and their roles in immune regulation, enabling detection, quantification, and functional analysis of ORAI1 in various experimental contexts.

What types of ORAI1 antibodies are available and how do they differ in their research applications?

Multiple ORAI1 antibodies are available targeting different epitopes of the protein:

These antibodies vary in their suitability for different applications, with some optimized for Western blotting at specific dilutions (e.g., 1:500-1:3000) , while others perform better in immunohistochemistry or flow cytometry applications. The choice of antibody should be guided by the specific experimental requirements and target epitope accessibility.

How should ORAI1 antibodies be validated before use in critical experiments?

Validation of ORAI1 antibodies should include multiple complementary approaches:

• Specificity testing:

  • Cross-reactivity assessment with ORAI2 and ORAI3

  • Competitive inhibition assays using various ORAI1 peptides (e.g., human ORAI1 Loop1/Loop2 peptides)

  • Testing in ORAI1 knockout/knockdown systems

• Application-specific validation:

  • For Western blotting: Verification of bands at both theoretical (32-33 kDa) and glycosylated (43-50 kDa) molecular weights

  • For IHC/ICC: Pattern consistency with known ORAI1 subcellular localization

  • For flow cytometry: Comparison with isotype controls and blocking peptides

• Functional validation:

  • Correlation of antibody binding with calcium influx inhibition

  • Effects on downstream signaling (e.g., NFAT activation)

  • Verification in human ORAI1 knock-in mouse models for translational studies

When validating antibodies, researchers should be aware that ORAI1 often migrates at higher than expected molecular weights in SDS-PAGE due to glycosylation and other post-translational modifications .

What are the optimal protocols for Western blotting detection of ORAI1?

For successful Western blot detection of ORAI1, researchers should follow these methodological guidelines:

• Sample preparation:

  • Use lysis buffers effective for membrane proteins (containing 1-2% detergent)

  • Include protease inhibitors to prevent degradation

  • Consider native vs. reducing conditions based on epitope accessibility

• Gel electrophoresis and transfer:

  • Use 10-12% polyacrylamide gels for optimal separation

  • Load positive control samples (e.g., A549 cells, A375 cells)

  • Transfer to PVDF membranes (preferred for hydrophobic proteins)

• Antibody incubation:

  • Block with 5% BSA in TBST (preferred for phosphoproteins)

  • Use recommended antibody dilutions (e.g., 1:500-1:3000)

  • Incubate primary antibody overnight at 4°C for optimal binding

• Data interpretation:

  • Expect bands at the theoretical molecular weight (32-33 kDa)

  • Look for additional bands at 43-50 kDa representing glycosylated forms

  • Validate band specificity using peptide competition or knockout samples

The selection of appropriate positive controls is critical; certain cell lines like A549 and A375 have been validated for ORAI1 expression and can serve as reliable Western blot controls .

How can ORAI1 antibodies be utilized in immunohistochemistry and immunocytochemistry applications?

For optimal IHC and ICC applications with ORAI1 antibodies:

• Fixation and permeabilization considerations:

  • For intracellular epitopes: 4% paraformaldehyde fixation followed by detergent permeabilization

  • For extracellular loop epitopes: Milder fixation to preserve native conformation

  • Optimize fixation time to balance epitope preservation and cellular architecture

• Antigen retrieval methods:

  • Test both heat-induced (citrate buffer, pH 6.0) and enzymatic methods

  • For paraffin sections, EDTA-based retrieval (pH 9.0) may provide better results

• Background reduction strategies:

  • Pre-absorption with non-specific proteins from the secondary antibody host

  • Include 0.1-0.3% Triton X-100 in antibody diluent for better penetration

  • Use fluorescent detection for lower background in co-localization studies

• Controls and validation:

  • Include tissue from ORAI1 knockout models or siRNA-treated cells

  • Perform peptide competition assays to confirm specificity

  • Compare staining patterns with known ORAI1 distribution in tissues/cells

When interpreting results, researchers should consider that ORAI1 distribution varies significantly between tissue types, with highest expression in immune cells, particularly T cells and mast cells .

What considerations are important when designing flow cytometry experiments with ORAI1 antibodies?

Flow cytometry with ORAI1 antibodies requires careful attention to:

• Cell preparation protocol:

  • Gentle dissociation methods to preserve membrane integrity

  • Appropriate fixation (2-4% paraformaldehyde) for intracellular epitopes

  • Optimization of permeabilization conditions (0.1% saponin or 0.1% Triton X-100)

• Antibody selection and staining:

  • Choose antibodies targeting extracellular loops for live cell staining

  • For intracellular epitopes, use thoroughly validated fixation-resistant clones

  • Consider using indirect staining to amplify signal for low-abundance ORAI1

• Panel design for multiparameter analysis:

  • Include markers for relevant cell populations (e.g., CD3 for T cells, CD117 and FcεRIα for mast cells)

  • Add functional markers (e.g., calcium flux indicators, activation markers)

  • Use appropriate compensation controls for multicolor experiments

• Analysis and interpretation:

  • Gate on relevant cell populations before analyzing ORAI1 expression

  • Compare with isotype controls and FMO (fluorescence minus one) controls

  • Correlate ORAI1 expression with functional parameters

For detecting low-level ORAI1 expression, signal amplification methods such as biotin-streptavidin systems may be employed, as demonstrated in research using biotin-conjugated anti-ORAI1 antibody with APC-conjugated streptavidin .

How can ORAI1 antibodies be used to study the interaction between ORAI1 and STIM1 in SOCE?

ORAI1 antibodies enable sophisticated analyses of ORAI1-STIM1 interactions:

• Co-immunoprecipitation approaches:

  • Use ORAI1 antibodies to pull down protein complexes

  • Detect co-precipitated STIM1 and associated signaling molecules

  • Compare interactions under store-depleted versus resting conditions

• Proximity-based interaction assays:

  • Proximity Ligation Assay (PLA) using antibodies against ORAI1 and STIM1

  • FRET/FLIM using antibody-conjugated fluorophores

  • Split complementation assays with antibody-guided reporters

• Super-resolution microscopy techniques:

  • STORM/PALM imaging using fluorescently-labeled antibodies

  • Track cluster formation and co-localization during store depletion

  • Quantify nanoscale spatial organization of signaling complexes

• Functional modulation studies:

  • Use function-blocking antibodies to disrupt specific interaction domains

  • Correlate structural changes with calcium influx measurements

  • Map critical interaction regions using domain-specific antibodies

Research has revealed that ORAI1 inactivation involves a calcium/cAMP signaling loop at the ORAI1 channel mouth, which shapes cellular Ca²⁺ signals and NFAT activation . Antibodies targeting specific regions of ORAI1 can help dissect these regulatory mechanisms and their functional consequences.

What role do post-translational modifications play in ORAI1 detection and function?

Post-translational modifications significantly impact ORAI1 detection and function:

• Glycosylation effects:

  • ORAI1 exists as the unmodified protein (32.7 kDa) and two major glycosylated forms (~43 kDa and ~50 kDa)

  • Glycosylation patterns vary by cell type and activation state

  • Deglycosylation treatments (PNGase F, Endo H) can help identify specific forms

• Phosphorylation considerations:

  • Phosphorylation affects channel gating and protein-protein interactions

  • Include phosphatase inhibitors in lysis buffers for consistent detection

  • Consider phospho-specific antibodies for studying regulatory events

• Impact on epitope accessibility:

  • Modifications may mask or expose antibody binding sites

  • Different antibodies may preferentially detect specific modified forms

  • Native versus denaturing conditions yield different results based on epitope exposure

• Functional correlations:

  • Track changes in modification patterns during cellular activation

  • Correlate modifications with channel activity and calcium signaling

  • Use site-specific mutants to validate modification-specific antibody detection

When interpreting Western blot results, researchers should expect heterogeneity in ORAI1 banding patterns due to these modifications and should validate their observations using appropriate controls and treatments.

How are ORAI1 antibodies being used to develop potential therapeutics for immune disorders?

ORAI1 antibodies show promising therapeutic applications in immune disorders:

• Development approaches:

  • Humanization of antibodies for clinical translation, as demonstrated with DS-2741a

  • Selection of function-blocking epitopes on extracellular domains

  • Engineering for optimal pharmacokinetics and tissue penetration

• Functional validation methods:

  • Inhibition of calcium influx in primary human T cells and mast cells

  • Suppression of cytokine production and degranulation responses

  • Assessment in human ORAI1 knock-in mouse models

• Disease model applications:

  • Demonstrated efficacy in house dust mite antigen-induced dermatitis models

  • Potential applications in other allergic and autoimmune conditions

  • Correlation between ORAI1 inhibition and disease modification

• Mechanism-of-action studies:

  • Competitive binding to extracellular loops to disrupt channel function

  • Interference with ORAI1-STIM1 coupling during store depletion

  • Inhibition of immune cell activation without complete immunosuppression

Research findings indicate that specific inhibition of ORAI1 represents a potential mechanism for treating atopic dermatitis and other immune diseases, as the humanized antibody DS-2741a demonstrated suppression of T-cell activation and mast cell degranulation in human ORAI1 knock-in mice .

Why does ORAI1 often appear at multiple molecular weights in Western blots?

ORAI1 typically appears at multiple molecular weights due to several factors:

• Glycosylation heterogeneity:

  • The theoretical molecular weight of unmodified ORAI1 is 32.7 kDa

  • Two major glycosylated forms appear at ~43 kDa and ~50 kDa

  • Glycosylation patterns vary by cell type and physiological state

• Other post-translational modifications:

  • Phosphorylation adds approximately 1 kDa per phosphate group

  • Ubiquitination or SUMOylation can significantly alter migration

  • Multiple modification combinations create pattern complexity

• Sample preparation influences:

  • Heat treatment duration affects membrane protein migration

  • Reducing agents can alter disulfide bond-dependent structures

  • Detergent selection impacts protein solubilization efficiency

• Technical considerations:

  • Gel percentage affects resolution of differently modified forms

  • Running conditions (voltage, time) influence band separation

  • Transfer efficiency varies for different molecular weight species

To confirm band identity, researchers should consider enzymatic deglycosylation treatments, comparison with ORAI1 knockout samples, and detection with multiple antibodies targeting different epitopes of the protein .

What controls are essential when working with ORAI1 antibodies?

Comprehensive control strategies for ORAI1 antibody experiments include:

For peptide competition assays, researchers can use various synthetic peptides corresponding to different regions of ORAI1, including human ORAI1 Loop1 peptide, human/cynomolgus monkey ORAI1 Loop2 peptide, and species orthologs like mouse Orai1 Loop2 peptide .

For confirming antibody specificity against ORAI homologs, it's essential to test against human ORAI2 Loop2 peptide and human ORAI3 Loop2 peptide, as some commercially available antibodies are specifically designed to have no cross-reactivity with ORAI2 or ORAI3 .

How can inconsistent results with ORAI1 antibodies be resolved?

To resolve inconsistent results with ORAI1 antibodies:

• Antibody validation refinement:

  • Verify antibody specificity using ORAI1 knockout systems

  • Test multiple antibodies targeting different epitopes

  • Confirm species cross-reactivity for your experimental model

• Protocol optimization steps:

  • Systematically test antibody dilutions (e.g., 1:500-1:3000 for Western blot)

  • Optimize incubation conditions (time, temperature, buffer composition)

  • Adjust blocking solutions to minimize background interference

• Sample preparation improvements:

  • Ensure complete lysis of membrane proteins with appropriate detergents

  • Include fresh protease/phosphatase inhibitors in all buffers

  • Standardize protein quantification and loading methods

• Application-specific troubleshooting:

  • For Western blotting: Optimize transfer conditions for membrane proteins

  • For IHC/ICC: Compare different fixation and antigen retrieval methods

  • For flow cytometry: Test alternative permeabilization protocols

• Data interpretation strategies:

  • Consider the impact of post-translational modifications on detection

  • Account for ORAI1 expression level differences between samples

  • Correlate protein detection with functional calcium influx measurements

As noted in several sources, ORAI1 antibody performance can be sample-dependent, and reagents should be titrated in each testing system to obtain optimal results .

How are ORAI1 antibodies contributing to our understanding of immune disorders?

ORAI1 antibodies are advancing immune disorder research through multiple approaches:

• SCID (Severe Combined Immunodeficiency) investigations:

  • ORAI1 homogenous deficiency in humans causes SCID with significantly impaired T-cell function

  • Antibodies enable analysis of mutant ORAI1 expression and localization

  • Correlation of structural defects with calcium signaling impairment

• Autoimmune disease mechanisms:

  • Assessment of ORAI1 expression and activation in patient samples

  • Analysis of ORAI1-dependent T cell hyperactivation pathways

  • Identification of potential therapeutic intervention points

• Allergic disease research:

  • Studies demonstrate that DS-2741a antibody suppresses T-cell activation and mast cell degranulation

  • This antibody ameliorated house dust mite antigen-induced dermatitis in human ORAI1 knock-in mice

  • Mechanistic insights into calcium-dependent allergic inflammation

• Therapeutic development platforms:

  • Function-blocking humanized antibodies as potential treatments

  • Correlation between epitope targeting and functional outcomes

  • Translation from animal models to human applications

The specific inhibition of ORAI1 has been identified as a potential mechanism for treating atopic dermatitis and other immune diseases, highlighting the therapeutic potential of antibodies targeting this calcium channel .

What new techniques are emerging for studying ORAI1 in calcium signaling microdomains?

Cutting-edge approaches for studying ORAI1 in calcium signaling microdomains include:

• Advanced imaging technologies:

  • Single-molecule localization microscopy with antibody fragments

  • Lattice light-sheet microscopy for 3D visualization of signaling domains

  • Correlative light and electron microscopy for ultrastructural context

• Molecular proximity analysis:

  • Engineered peroxidase-antibody conjugates for proximity labeling

  • Mass spectrometry identification of microdomain components

  • Optogenetic manipulation of ORAI1-containing complexes

• Functional microdomain mapping:

  • Local calcium uncaging combined with antibody-based detection

  • Subcellular optogenetic activation of specific signaling nodes

  • Correlation between microdomain organization and calcium signatures

• Computational modeling integration:

  • Spatiotemporal simulation of antibody-defined ORAI1 clusters

  • Machine learning analysis of microdomain organizational patterns

  • Multi-scale models connecting molecular interactions to cellular responses

Research has revealed that CRAC channels assemble in Ca²⁺ signaling microdomains where Ca²⁺ influx is coupled to calmodulin and calcineurin signaling, activating NFAT transcription factors recruited to ORAI1 via AKAP5 . These complex signaling hubs can now be systematically mapped using advanced antibody-based technologies.

How might ORAI1 antibodies be used to distinguish the roles of different ORAI family members?

ORAI1 antibodies enable sophisticated discrimination between ORAI family members:

• Comparative expression analysis:

  • Tissue distribution mapping shows ORAI1 mainly in immune cells, ORAI2 primarily in brain, lungs, spleen, and small intestine, and ORAI3 abundant in many solid organs

  • Antibodies with verified lack of cross-reactivity provide accurate expression profiles

  • Correlation of expression patterns with tissue-specific calcium signaling

• Functional discrimination approaches:

  • Selective functional blocking of ORAI1 reveals its specific contributions

  • Comparison with ORAI2/ORAI3 knockdown phenotypes

  • Analysis of calcium signature differences (ORAI1 for sustained plateaus vs. ORAI2/ORAI3 for oscillatory patterns)

• Complex formation analysis:

  • Immunoprecipitation with ORAI1-specific antibodies to identify heteromeric channels

  • Quantification of ORAI1:ORAI2:ORAI3 stoichiometry in different tissues

  • Correlation between subunit composition and channel properties

• Therapeutic targeting implications:

  • Selective ORAI1 inhibition without affecting ORAI2/ORAI3 function

  • Tissue-specific targeting based on differential expression

  • Minimization of off-target effects through isoform-specific antibodies

The availability of highly specific antibodies that recognize ORAI1 without cross-reactivity to ORAI2 or ORAI3 provides powerful tools for distinguishing the unique contributions of each family member to calcium signaling in different physiological and pathological contexts.