CRACR2A Antibody
Description
Introduction to CRACR2A Antibody
CRACR2A antibodies are immunological reagents specifically designed to detect the Calcium Release-Activated Calcium Channel Regulator 2A (CRACR2A) protein, also known as EF-hand calcium-binding domain-containing protein 4B (EFCAB4B) . These antibodies serve as crucial tools for investigating the expression, localization, and functional aspects of CRACR2A in both normal physiology and pathological conditions. The target protein, CRACR2A, is a calcium-binding protein that plays a key role in store-operated calcium entry (SOCE) in T-cells by regulating CRAC channel activation .
The development of specific CRACR2A antibodies has enabled significant advancements in understanding this protein's role in various cellular processes, particularly in immune cell function. By providing means to detect and study CRACR2A, these antibodies contribute to our growing knowledge of calcium signaling pathways and their implications in immune disorders, making them invaluable resources in immunological and cell biology research.
Types and Characteristics of CRACR2A Antibodies
CRACR2A antibodies are available in several formats with distinct characteristics suited for different research applications. The diversity in these antibodies provides researchers flexibility in experimental design and analysis.
Antibody Classifications
Based on their production method and specificity, CRACR2A antibodies can be categorized as:
-
Monoclonal Antibodies: These include products like the CRACR2A Monoclonal Antibody (clone 4C1F5) conjugated with CoraLite® Plus 488 . Monoclonal antibodies offer high specificity for particular epitopes of the CRACR2A protein, ensuring consistent results across experiments.
-
Polyclonal Antibodies: Products such as the CRACR2A Polyclonal Antibody (15206-1-AP) provide broader epitope coverage, potentially increasing detection sensitivity . Boster Bio's Anti-EFCAB4B CRACR2A Antibody is another example of a polyclonal antibody raised against a synthetic peptide near the carboxy terminus of human EFCAB4B .
-
Recombinant Antibodies: Creative Biolabs offers Mouse Anti-CRACR2A Recombinant Antibody (clone CB64YJ), which combines high specificity with reproducible production methods .
Host Species and Applications
CRACR2A antibodies are produced in various host species and validated for multiple applications:
-
Host Species: Common hosts include rabbit and mouse, allowing flexibility in experimental design, particularly for co-staining applications .
-
Applications: These antibodies are validated for techniques including:
-
Species Reactivity: Available antibodies show reactivity with samples from various species, including human, mouse, and non-human primates .
The table below compares key characteristics of selected CRACR2A antibodies based on the available data:
Structure and Function of the CRACR2A Target Protein
Understanding the structure and function of CRACR2A provides essential context for appreciating the significance and applications of CRACR2A antibodies in research.
Protein Structure
CRACR2A exists in multiple isoforms with distinct structural characteristics:
-
Isoform Diversity:
-
Structural Domains:
Molecular Function
CRACR2A performs several critical cellular functions:
-
Calcium Signaling Regulation:
-
Dynamic Response to Calcium:
-
Additional Functions:
Research Applications of CRACR2A Antibodies
CRACR2A antibodies are utilized in diverse research applications, facilitating investigations into calcium signaling pathways and immune cell function.
Detection and Localization Studies
-
Protein Expression Analysis:
-
Subcellular Localization:
Functional Studies
-
Calcium Signaling Research:
-
Investigation of CRACR2A's role in store-operated calcium entry
-
Analysis of calcium flux in immune cells using fluorescent calcium indicators alongside CRACR2A antibodies
-
-
Protein-Protein Interaction Studies:
-
Immunoprecipitation to identify and characterize CRACR2A binding partners
-
Proximity ligation assays to verify interactions in situ
-
-
T-cell Function Analysis:
Clinical Research Applications
-
Immunodeficiency Studies:
-
Inflammation Research:
CRACR2A in Immune Cell Function
Research utilizing CRACR2A antibodies has revealed critical roles for this protein in immune cell function, particularly in T-cells and neutrophils.
T-cell Function
CRACR2A plays essential roles in T-cell activation and function:
-
Calcium Signaling: Regulates store-operated calcium entry (SOCE), a critical process for T-cell activation
-
T-cell Receptor (TCR) Signaling: Contributes to proximal TCR signaling pathways, including JNK phosphorylation
-
Vesicle Trafficking: The longer CRACR2A isoform localizes to vesicles that translocate toward the immunological synapse during T-cell activation
-
Cytokine Production: Supports T-cell proliferation and cytokine secretion, critical functions for effective immune responses
Neutrophil Function
Recent research has identified important roles for CRACR2A in neutrophil biology:
-
Neutrophil Recruitment: Promotes neutrophil recruitment to sites of sterile inflammation, such as in ischemic stroke
-
Adhesion and Migration: Facilitates neutrophil adhesive and migratory functions by enhancing calcium mobilization and β2 integrin activation
-
Molecular Mechanisms: Rapidly interacts with Stim1 (stromal interaction molecule 1) after agonist stimulation, facilitating calcium mobilization and increasing the ligand-binding function of β2 integrin
-
Experimental Evidence: Studies using myeloid-specific Cracr2a conditional knockout mice showed significantly reduced adhesion, crawling, and transmigration of neutrophils on ear and cremaster venules in tumor necrosis factor-α-induced sterile inflammation compared to wild-type controls
Clinical Relevance and Pathological Implications
CRACR2A antibodies have facilitated discoveries regarding the clinical relevance of this protein, particularly in immunological disorders.
Primary Immunodeficiency
A groundbreaking study using CRACR2A antibodies identified biallelic mutations in CRACR2A as a novel cause of primary immunodeficiency:
-
Case Study Findings: A 33-year-old patient of East-Asian origin with compound heterozygous mutations in CRACR2A exhibited:
-
Cellular Analysis: Using CRACR2A antibodies, researchers demonstrated:
-
Longitudinal Immune Monitoring: The table below presents immune cell counts from the affected patient over time:
| Parameter | 2005 | 2011 | 2017 | 2019 | 2021 (Feb) | 2021 (Nov) | Reference Range |
|---|---|---|---|---|---|---|---|
| Total lymphocyte count | 2,880 | 990 | 1,201 | 1,572 | 1,472 | 1,268 | 1000–2800 cells/µl |
| CD3+ T cells | 2,505 | 733 | 891 | 990 | 952 | 846 | 700–1200 cells/µl |
| CD4+ T cells | 216 | 235 | 148 | 165 | 122 | 112 | 300–1400 cells/µl |
| CD19+ B cells | 245 | 102 | 73 | 77 | 87 | 71 | 100–500 cells/µl |
Source: Data extracted from publication on biallelic mutations in CRACR2A
Inflammatory Conditions
CRACR2A antibodies have enabled research demonstrating this protein's role in inflammatory processes:
-
Sterile Inflammation: CRACR2A promotes neutrophil recruitment to sites of sterile inflammation, such as occurs in ischemic stroke
-
Therapeutic Potential: Research suggests that targeting the Stim1-CRACR2A interaction could modulate neutrophil recruitment, potentially offering therapeutic approaches for inflammatory conditions
Technical Considerations for CRACR2A Antibody Applications
Researchers working with CRACR2A antibodies should consider several technical aspects to optimize experimental outcomes.
Antibody Selection
When selecting a CRACR2A antibody, researchers should consider:
-
Isoform Recognition: Some antibodies recognize both CRACR2A-c and CRACR2A-a isoforms, while others may be specific to one isoform
-
Epitope Mapping: Understanding the epitope recognized by the antibody is crucial, especially when studying specific domains or mutant forms of CRACR2A
-
Application Suitability: Different antibodies may perform optimally in specific applications; validated applications should be confirmed before experimental use
Experimental Protocols
Optimal protocols for CRACR2A antibody applications include:
-
Western Blotting:
-
Immunohistochemistry:
-
Effective antigen retrieval methods
-
Appropriate blocking to minimize background signal
-
-
Immunofluorescence:
-
Co-staining with markers of subcellular compartments to determine precise localization
-
Appropriate fixation and permeabilization protocols
-
Validation Approaches
Robust validation of CRACR2A antibody specificity can be achieved through:
-
Knockout/Knockdown Controls: Using CRACR2A-deficient samples (e.g., CRISPR/Cas9 knockout cell lines) as negative controls
-
Overexpression Systems: Complementary use of CRACR2A-overexpressing cells as positive controls
-
Peptide Competition: Using immunizing peptides to confirm signal specificity
Future Directions in CRACR2A Antibody Research
The field of CRACR2A antibody research continues to evolve, with several promising future directions:
Advanced Antibody Development
-
Isoform-Specific Antibodies: Development of antibodies that specifically recognize either CRACR2A-c or CRACR2A-a isoforms to better understand their distinct functions
-
Domain-Specific Antibodies: Creation of antibodies targeting specific functional domains (e.g., EF-hand, coiled-coil, or GTPase domains) to dissect domain-specific functions
-
Phospho-Specific Antibodies: Development of antibodies recognizing phosphorylated forms of CRACR2A to study its post-translational regulation
Emerging Applications
-
Live-Cell Imaging: Development of non-disruptive antibody-based probes for tracking CRACR2A dynamics in living cells
-
Therapeutic Targeting: Exploration of antibody-based approaches to modulate CRACR2A function in inflammatory conditions or immunodeficiencies
-
Diagnostic Development: Potential use of CRACR2A antibodies in diagnostic assays for CRACR2A-associated immunodeficiencies
Product Specs
Target Background
CRACR2A is also a Rab GTPase that mediates the trafficking of Weibel-Palade bodies (WPBs) to the microtubule organizing center (MTOC) in endothelial cells in response to acute inflammatory stimuli. During histamine (but not thrombin) stimulation of endothelial cells, the dynein-bound form of CRACR2A induces retrograde transport of a subset of WPBs along microtubules to the MTOC in a Ca(2+)-independent manner. Its GTPase activity is essential for this function.
As a Ca(2+)-regulated dynein adapter protein, CRACR2A activates dynein-mediated transport and dynein-dynactin motility on microtubules, regulating the endosomal trafficking of CD47. It acts as an intracellular signaling module that bridges two important T-cell receptor (TCR) signaling pathways, Ca(2+)-NFAT and JNK, to affect T-cell activation. In resting T-cells, CRACR2A is predominantly localized near the trans-Golgi network in a GTP-bound form. Upon TCR stimulation, it localizes at the immunological synapse through interaction with VAV1, activating downstream Ca(2+)-NFAT and JNK signaling pathways. CRACR2A plays a role in T-helper 1 (Th1) cell differentiation and T-helper 17 (Th17) cell effector function, and contributes to store-operated Ca(2+) entry (SOCE) in T-cells by regulating CRAC channel activation.
- Research has characterized CRACR2A protein, which encodes a large Rab GTPase with multiple functional domains, unlike smaller Rab GTPases. This study revealed its unexpected role in regulating intracellular signaling pathways critical for T cell activation. PMID: 27221160
- GTP binding and prenylation of CRACR2A were found to be associated with its localization near the Golgi and its stability. PMID: 27016526
- Endothelial cells contain a long variant of CRACR2A, an EF-hand-containing Rab protein that does not affect CRAC channels. PMID: 25475730
- CRACR2A interacts directly with Orai1 and STIM1, forming a ternary complex that dissociates at elevated Ca(2+) concentrations. It is a key regulator of CRAC channel-mediated SOCE. PMID: 20418871
Q&A
What is CRACR2A and why are antibodies against it important in immunological research?
CRACR2A is a large Rab GTPase abundantly expressed in T cells that functions as a signal transmitter between T cell receptor stimulation and activation of the Ca²⁺-NFAT and JNK-AP1 pathways . It contains two predicted EF-hand motifs in its N-terminus and a coiled-coil domain with leucine-rich regions . Antibodies against CRACR2A are crucial for studying its role in immune cell function, particularly since mutations in CRACR2A have been linked to combined immunodeficiency disorders and various inflammatory conditions .
To effectively use CRACR2A antibodies in your research, consider these methodological approaches:
-
Use antibodies targeting different epitopes of CRACR2A to distinguish between full-length and truncated variants
-
Include appropriate controls when investigating CRACR2A expression, especially when studying patient samples with potential mutations
-
Validate antibody specificity using CRACR2A knockout cells, as demonstrated in studies using CRISPR/Cas9-mediated gene deletion
What experimental techniques can CRACR2A antibodies be reliably used for?
Based on published research, CRACR2A antibodies have been successfully employed in:
When selecting a technique, consider the specific question you're addressing and the nature of your samples (cell lines, primary cells, or patient samples).
How can I verify the specificity of a CRACR2A antibody?
Antibody specificity is crucial for reliable results. To validate CRACR2A antibodies:
-
Use CRACR2A knockout cells as negative controls, which can be generated using CRISPR/Cas9 (sgRNA targets have been validated in published studies)
-
Perform siRNA-mediated knockdown of CRACR2A to confirm signal reduction
-
Test for cross-reactivity with the related protein CRACR2B, especially in HEK293 cells where CRACR2B appears to have stronger effects on SOCE than CRACR2A
-
Confirm expected molecular weight patterns: ~90 kDa for wild-type CRACR2A and ~30 kDa for truncated variants like R144G/E300*
-
Include recombinant CRACR2A protein as a positive control when available
What are the key differences between detecting CRACR2A in T cells versus neutrophils?
Recent research has revealed important differences in CRACR2A function between cell types:
-
T cells: CRACR2A is abundantly expressed and critical for T cell receptor signaling, Ca²⁺ mobilization, and cytokine production. Mutations in CRACR2A can lead to reduced SOCE and impaired cytokine production (IFN-γ, IL-2, TNF) .
-
Neutrophils: CRACR2A promotes neutrophil adhesive and migratory functions by facilitating Ca²⁺ mobilization and β2 integrin activation. It plays a significant role in neutrophil recruitment during sterile inflammation .
When working with neutrophils, consider using myeloid-specific conditional knockout mouse models as controls, as these have been validated in intravital microscopy studies of neutrophil recruitment .
How can CRACR2A antibodies be used to investigate the ternary complex formation with Orai1 and STIM1?
CRACR2A directly interacts with both Orai1 and STIM1 to form a ternary complex critical for CRAC channel activation . To study this complex:
-
Perform co-immunoprecipitation experiments using CRACR2A antibodies in cells expressing tagged versions of Orai1 and STIM1
-
Compare binding under resting conditions versus after store depletion (e.g., with thapsigargin treatment)
-
Use Ca²⁺ chelators to test the Ca²⁺-dependency of the interactions, as CRACR2A dissociates from Orai1 and STIM1 at higher Ca²⁺ concentrations
-
Consider using proximity ligation assays to visualize the interactions in intact cells
-
For advanced structural studies, use antibodies to confirm the integrity of protein complexes before crystallization or cryo-EM analysis
Research has shown enhanced binding between CRACR2A, Orai1, and STIM1 upon store depletion, making this a critical consideration in experimental design .
What approaches can I use to detect mutant forms of CRACR2A in patient samples?
When studying patient samples with CRACR2A mutations (like those described in the literature: E278D, R144G, E300*), consider:
-
Using antibodies targeting different regions of CRACR2A to detect both full-length and truncated proteins
-
Performing quantitative RT-PCR to measure transcript levels, as some mutations may affect mRNA stability
-
Combining protein and mRNA analysis, as patient T cells have shown pronounced reduction in CRACR2A protein levels despite detectable transcripts
-
Including functional assays to correlate antibody detection with phenotypic effects:
For the truncated double mutant (R144G/E300*), note that expression levels may be much lower than wild-type or E278D mutant proteins, suggesting transcript instability .
How can CRACR2A antibodies be employed to study Ca²⁺ signaling dynamics in immune cells?
CRACR2A functions as a cytosolic Ca²⁺ sensor and regulates store-operated calcium entry (SOCE) . To investigate its role in Ca²⁺ signaling:
-
Combine CRACR2A immunostaining with Ca²⁺ imaging techniques to correlate protein localization with Ca²⁺ flux
-
Use EF-hand mutants of CRACR2A as controls, as these enhance STIM1 clustering and elevate cytoplasmic Ca²⁺
-
Study the temporal dynamics of CRACR2A-Stim1 interaction after agonist stimulation in different cell types:
-
In T cells: After TCR stimulation
-
In neutrophils: After inflammatory stimuli
-
-
Compare wild-type cells with CRACR2A-deficient cells to assess:
Research has shown that CRACR2A rapidly interacts with Stim1 after agonist stimulation to facilitate Ca²⁺ mobilization, increasing the ligand-binding function of β2 integrin in neutrophils .
What methodological considerations are important when using CRACR2A antibodies in intravital microscopy studies?
Recent research has employed four-dimensional confocal intravital microscopy to study the role of neutrophil CRACR2A in vivo . When using CRACR2A antibodies in such studies:
-
Use myeloid-specific Cracr2a conditional knockout mice as controls
-
Consider labeling strategies that allow single-cell behavioral analysis
-
For neutrophil recruitment studies, examine multiple parameters:
-
Adhesion to vascular endothelium
-
Crawling behavior
-
Transmigration through vessel walls
-
-
Compare findings across different inflammation models:
-
TNF-α-induced sterile inflammation
-
Focal brain ischemia models
-
Other tissue-specific inflammation models
-
Researchers have shown that Cracr2a conditional knockout mice exhibit significantly reduced adhesion, crawling, and transmigration of neutrophils on ear and cremaster venules in TNF-α-induced sterile inflammation .
How can I use CRACR2A antibodies to validate gene editing experiments and knockout models?
When validating CRACR2A knockout models:
-
Use Western blotting with CRACR2A antibodies to confirm protein deletion
-
Include analysis of multiple cell types, as CRACR2A function differs between T cells and neutrophils
-
Validate functional consequences of knockout through:
-
Consider rescue experiments by re-expressing CRACR2A to confirm phenotype specificity
-
Check for compensatory changes in related proteins (e.g., CRACR2B, ORAI1, STIM1)
Research has shown that sgRNA#1 and sgRNA#2 targeting CRACR2A achieved efficient deletion in Jurkat T cells, which can serve as reference points for your own knockout strategies .
What novel applications of CRACR2A antibodies might advance our understanding of inflammatory diseases?
Given the associations between CRACR2A SNPs and cardiovascular or inflammatory diseases , future research could:
-
Use CRACR2A antibodies to study protein expression and function in:
-
Ischemic stroke models
-
Cardiovascular inflammation
-
Other sterile inflammatory conditions
-
-
Develop tissue-specific approaches to detect CRACR2A activity in different organs
-
Investigate CRACR2A expression in patient cohorts with inflammatory conditions
-
Explore the therapeutic potential of targeting CRACR2A with blocking peptides:
How might CRACR2A antibodies contribute to understanding the mechanisms behind CRACR2A-associated immunodeficiency?
Building on the identification of CRACR2A variants in late-onset combined immunodeficiency :
-
Develop antibodies specific to common CRACR2A mutations to facilitate screening
-
Use antibodies in conjunction with functional assays to create diagnostic workflows
-
Study the differential effects of CRACR2A mutations on:
-
Investigate potential connections between CRACR2A dysfunction and other primary immunodeficiencies
-
Explore compensatory mechanisms in different immune cell types that might explain the late onset of symptoms
CRACR2A deficiency has been proposed for inclusion within the group of combined immunodeficiency disorders in the IEI classification, highlighting its clinical significance .
