cracr2b Antibody

What is CRACR2B and what cellular functions does it serve?

CRACR2B (also known as EFCAB4A) is a calcium-binding protein involved in calcium signaling pathways. It shares structural similarities with CRACR2A, which contains calcium-binding EF-hand domains, protein-interacting coiled-coil domains, and a C-terminal Rab GTPase domain in its longer isoform . CRACR2A interacts with ORAI1 and STIM1 proteins to regulate store-operated calcium entry (SOCE), which is a critical process in calcium homeostasis and signaling across various cell types . While CRACR2B functions are less extensively documented than CRACR2A, its homology suggests similar roles in calcium regulation pathways. CRACR2A has been shown to interact with Vav1 and regulate JNK phosphorylation, with its GTPase domain and prenylation necessary for this role . Understanding these molecular interactions provides context for researchers using CRACR2B antibodies in signaling pathway investigations.

What types of CRACR2B antibodies are available for research applications?

The primary types of CRACR2B antibodies available for research include rabbit polyclonal antibodies against human CRACR2B. Specific examples include:

• Rabbit Polyclonal Anti-CRACR2B Antibody (HPA046217), validated for immunohistochemistry (IHC) and Western blot (WB) applications with enhanced validation protocols .

• Rabbit polyclonal antibodies raised against synthetic peptides of EFCAB4A (another name for CRACR2B), which are recommended for Western blot applications at dilutions of 1-2 μg/ml .

These antibodies are unconjugated (not linked to reporter molecules) and designed to recognize specific epitopes of the human CRACR2B protein . The polyclonal nature of these antibodies means they recognize multiple epitopes on the target protein, potentially providing stronger signals but with some risk of increased background compared to monoclonal antibodies.

What validation methods ensure CRACR2B antibody specificity and reliability?

Rigorous validation of CRACR2B antibodies involves multiple methodologies:

• Immunohistochemistry (IHC) validation: Antibodies are tested on tissue sections to confirm specific cellular and subcellular staining patterns consistent with known CRACR2B expression .

• Western blot validation: Antibodies should detect bands of predicted molecular weight for CRACR2B in appropriate cell or tissue lysates .

• Enhanced validation: More comprehensive testing may include genetic approaches (using knockout or knockdown models), independent antibody verification (using antibodies targeting different epitopes), and recombinant expression systems .

• Orthogonal validation: Correlating protein detection with mRNA expression data from the same samples, as demonstrated in studies of the related protein CRACR2A where both immunoblotting and qRT-PCR were employed .

These validation approaches collectively establish the specificity, sensitivity, and reproducibility of CRACR2B antibodies across different experimental contexts. Researchers should review available validation data when selecting antibodies for their specific applications.

How might CRACR2B antibodies help elucidate calcium signaling defects in immunodeficiency?

CRACR2B antibodies can serve as critical tools in investigating calcium signaling defects in immunodeficiency, drawing on insights from related CRACR2A research. Studies have shown that biallelic mutations in CRACR2A lead to combined immunodeficiency, characterized by recurrent infections, panhypogammaglobulinemia, and CD4+ T cell lymphopenia . CRACR2B antibodies can help researchers:

• Quantify protein expression levels: As demonstrated with CRACR2A, immunoblotting can reveal reduced protein levels in patient cells compared to healthy controls .

• Investigate signaling cascade disruptions: CRACR2A mutations affect store-operated calcium entry (SOCE) and JNK phosphorylation, impairing T cell proliferation and cytokine production . Similar approaches with CRACR2B antibodies might reveal related or distinct signaling defects.

• Analyze protein-protein interactions: Co-immunoprecipitation with CRACR2B antibodies could identify altered interactions with binding partners like ORAI1, STIM1, or other signaling molecules in disease states.

• Track subcellular localization: Immunofluorescence with CRACR2B antibodies can reveal changes in protein localization that might contribute to signaling defects.

By employing CRACR2B antibodies in these applications, researchers can build comprehensive models of calcium signaling disruptions in immunodeficiency and potentially identify novel therapeutic targets.

What approaches should researchers use when studying the relationship between CRACR2B and T cell function?

When investigating CRACR2B's role in T cell function, researchers should employ multifaceted approaches:

• Expression analysis across T cell subsets: Quantify CRACR2B expression in different T cell populations (naïve, memory, effector, regulatory) using flow cytometry or immunoblotting with CRACR2B antibodies .

• T cell activation studies: Compare CRACR2B levels and localization before and after TCR stimulation using anti-CD3/CD28 or PMA/ionomycin, as performed with CRACR2A .

• Calcium flux measurement: Correlate CRACR2B expression with calcium flux in T cells using calcium-sensitive dyes while manipulating CRACR2B levels.

• Functional assays: Assess how CRACR2B knockdown or overexpression affects T cell proliferation, cytokine production, and cell survival, similar to approaches used for CRACR2A research .

• Mutational analysis: Express CRACR2B mutants in T cells and evaluate functional consequences, as demonstrated with CRACR2A E278D and R144G/E300* mutants .

These approaches collectively provide a comprehensive assessment of CRACR2B's functional significance in T cell biology and potential contributions to immunological disorders.

How do computational methods complement antibody-based detection of CRACR2B in structural studies?

Computational methods provide valuable complementary approaches to antibody-based CRACR2B detection:

• Epitope prediction algorithms: Computational tools can predict antigenic determinants on CRACR2B, informing antibody selection for specific domains (e.g., EF-hand, coiled-coil, or GTPase domains) .

• Structural modeling: Homology modeling based on related proteins like CRACR2A can predict CRACR2B's tertiary structure and potential binding interfaces, guiding antibody epitope selection .

• Binding free energy calculations: Computational alanine scanning methods, similar to those described by Robin et al., can predict key residues contributing to antibody-antigen binding energy . This helps researchers understand which amino acids are critical for recognition by specific antibodies.

• Paratope-epitope mapping: Tools like Paratome and proABC can computationally predict antibody binding regions (ABRs) and their interaction with CRACR2B epitopes, providing insights beyond traditional complementarity-determining region (CDR) definitions .

• Molecular dynamics simulations: These can model the dynamic interactions between antibodies and CRACR2B under different conditions, predicting conformational changes that might affect epitope accessibility.

By integrating these computational approaches with experimental antibody-based detection, researchers can gain deeper insights into CRACR2B structure-function relationships and optimize detection strategies for different experimental contexts.

What are the optimal protocols for using CRACR2B antibodies in Western blotting?

For optimal Western blot detection of CRACR2B using antibodies, researchers should follow these methodological guidelines:

• Sample preparation:

  • Extract proteins using RIPA buffer supplemented with protease and phosphatase inhibitors

  • Quantify protein concentrations using BCA or Bradford assays to ensure equal loading

  • Denature samples in Laemmli buffer containing DTT or β-mercaptoethanol at 95°C for 5 minutes

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal resolution of CRACR2B

  • Transfer to PVDF membranes at 100V for 60-90 minutes in cold transfer buffer with 20% methanol

  • Confirm transfer efficiency with Ponceau S staining

• Antibody incubation:

  • Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

  • Incubate with primary anti-CRACR2B antibody at recommended dilutions (typically 1-2 μg/ml) overnight at 4°C

  • Wash thoroughly with TBST (4 × 5 minutes)

  • Incubate with appropriate HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

  • Wash again with TBST (4 × 5 minutes)

• Detection and validation:

  • Develop using enhanced chemiluminescence (ECL) reagents

  • Include appropriate positive controls (e.g., cells known to express CRACR2B)

  • Use β-actin as a loading control, as demonstrated in CRACR2A studies

  • Consider additional validation by comparing unstimulated versus stimulated samples (e.g., PMA/ionomycin)

Following these protocols will maximize specificity and minimize background when detecting CRACR2B via Western blotting, enabling accurate quantification of protein expression across experimental conditions.

What considerations are important when using CRACR2B antibodies for immunohistochemistry?

When employing CRACR2B antibodies for immunohistochemistry (IHC), researchers should address several critical methodological considerations:

• Tissue preparation and fixation:

  • Optimal fixation with 10% neutral-buffered formalin for 24 hours preserves antigenicity

  • Paraffin embedding and sectioning at 4-5 μm thickness provides suitable morphology

  • Consider testing both frozen and paraffin-embedded samples if antigen accessibility is a concern

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Determine optimal retrieval time (typically 15-30 minutes) to balance antigen retrieval with tissue integrity

  • Validated anti-CRACR2B antibodies like HPA046217 have established antigen retrieval protocols

• Antibody dilution and incubation:

  • Titrate antibody concentrations to determine optimal dilution that maximizes specific signal while minimizing background

  • Incubate sections with primary antibody overnight at 4°C or for 1-2 hours at room temperature

  • Use appropriate detection systems (e.g., polymer-based HRP systems) with chromogenic substrates like DAB

• Controls and counterstaining:

  • Include positive control tissues with known CRACR2B expression

  • Implement negative controls (primary antibody omission, isotype controls)

  • Use hematoxylin counterstaining for nuclear visualization

  • Consider dual staining with markers of specific cell types when investigating tissue distribution

• Interpretation guidelines:

  • Evaluate both staining intensity and pattern (membranous, cytoplasmic, nuclear)

  • Document subcellular localization, which may provide insights into functional status

  • Quantify expression using established scoring systems if performing comparative studies

These methodological considerations will enable researchers to generate reliable and reproducible IHC data when investigating CRACR2B expression across different tissues and experimental conditions.

How can CRACR2B antibodies be employed in studies of protein-protein interactions?

CRACR2B antibodies can be valuable tools in investigating protein-protein interactions through several methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Lyse cells in non-denaturing buffers containing mild detergents (e.g., NP-40, Triton X-100)

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Incubate cleared lysates with anti-CRACR2B antibodies overnight at 4°C

  • Capture antibody-protein complexes with protein A/G beads

  • Wash stringently to remove non-specific interactions

  • Elute and analyze interacting partners by immunoblotting or mass spectrometry

  • Based on CRACR2A studies, potential interacting partners might include ORAI1, STIM1, and components of the JNK signaling pathway

• Proximity ligation assay (PLA):

  • Fix cells and perform permeabilization

  • Incubate with primary antibodies against CRACR2B and potential interacting partners

  • Apply PLA probes (oligonucleotide-linked secondary antibodies)

  • Perform ligation and amplification reactions

  • Visualize interaction signals as fluorescent spots using confocal microscopy

  • This technique offers single-molecule resolution of interactions in their native cellular context

• Immunofluorescence co-localization:

  • Perform double immunostaining with antibodies against CRACR2B and potential binding partners

  • Use differentially labeled secondary antibodies (e.g., Alexa Fluor 488 and 594)

  • Analyze co-localization using confocal microscopy and quantitative co-localization metrics

  • This approach can reveal spatial relationships that suggest functional interactions

• FRET (Förster Resonance Energy Transfer):

  • Label anti-CRACR2B and partner protein antibodies with donor-acceptor fluorophore pairs

  • Measure energy transfer as an indicator of molecular proximity (<10 nm)

  • This technique can confirm direct interactions between CRACR2B and binding partners

These methodological approaches allow researchers to characterize CRACR2B's interactome and elucidate its role in calcium signaling and other cellular pathways, potentially revealing novel therapeutic targets for immunological disorders.

How should researchers address non-specific binding when using CRACR2B antibodies?

Non-specific binding is a common challenge when working with antibodies, including those targeting CRACR2B. Researchers can implement several methodological solutions:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, non-fat dry milk, normal serum, commercial blockers)

  • Increase blocking time (from 1 hour to overnight)

  • Use blocking agents that match the species in which the secondary antibody was raised

• Adjust antibody concentrations and incubation conditions:

  • Titrate primary antibody to find the minimum concentration that produces specific signal

  • Reduce incubation temperature (from room temperature to 4°C)

  • Add 0.1-0.5% Tween-20 or Triton X-100 to reduce hydrophobic interactions

  • Consider increasing salt concentration in wash buffers to disrupt low-affinity interactions

• Implement additional specificity controls:

  • Use blocking peptides specific to the CRACR2B epitope

  • Test the antibody on samples with known CRACR2B expression levels

  • Include negative controls (CRACR2B knockout or knockdown samples)

  • Validate with multiple antibodies targeting different CRACR2B epitopes

• Pre-adsorb antibodies if cross-reactivity is suspected:

  • Incubate antibodies with proteins/tissues that potentially cause cross-reactivity

  • Use the pre-adsorbed antibody for the actual experiment to reduce background

• Consider alternative detection methods:

  • Switch from chromogenic to fluorescent detection for reduced background

  • Use detection systems with enhanced sensitivity and specificity

  • Employ signal amplification methods only when necessary, as they can increase background

By systematically addressing these aspects, researchers can significantly improve signal-to-noise ratios and ensure reliable detection of CRACR2B across different experimental platforms.

What strategies help resolve discrepancies between mRNA and protein expression data for CRACR2B?

Discrepancies between CRACR2B mRNA and protein levels are common in research and require systematic troubleshooting approaches:

• Technical validation:

  • Confirm primer and antibody specificity through appropriate controls

  • Use multiple primer pairs targeting different regions of CRACR2B mRNA

  • Test different CRACR2B antibodies recognizing distinct epitopes

  • Employ absolute quantification methods for both mRNA (digital PCR) and protein (quantitative Western blotting)

• Temporal considerations:

  • Examine time-course experiments to account for delays between transcription and translation

  • Compare mRNA and protein half-lives, as demonstrated in studies where both RT-PCR and immunoblotting were used to track expression dynamics

  • Consider circadian or cell-cycle-dependent regulation

• Post-transcriptional regulation assessment:

  • Investigate microRNA regulation of CRACR2B mRNA

  • Examine alternative splicing events that might generate protein isoforms not detected by certain antibodies

  • Assess RNA stability factors that might affect CRACR2B mRNA turnover

• Post-translational modification analysis:

  • Evaluate protein degradation rates through cycloheximide chase experiments

  • Investigate ubiquitination and proteasomal degradation pathways

  • Examine other post-translational modifications that might affect antibody epitope recognition

• Subcellular localization and extraction efficiency:

  • Ensure extraction protocols efficiently recover CRACR2B from all cellular compartments

  • Compare cytoplasmic versus membrane-associated protein pools

  • Consider cell fractionation approaches to track protein distribution

By systematically addressing these potential sources of discrepancy, researchers can develop a more comprehensive understanding of CRACR2B regulation and avoid misinterpretation of experimental results.

How can researchers differentiate between CRACR2A and CRACR2B when using antibodies with potential cross-reactivity?

Differentiating between the related proteins CRACR2A and CRACR2B requires careful methodological approaches to ensure specific detection:

• Epitope selection and antibody validation:

  • Choose antibodies targeting regions with minimal sequence homology between CRACR2A and CRACR2B

  • Request epitope information from antibody manufacturers to assess potential cross-reactivity

  • Perform BLAST analysis of antibody epitopes against both proteins to predict specificity

  • Validate antibodies using overexpression systems with tagged CRACR2A and CRACR2B constructs

• Controlled expression systems:

  • Test antibodies on samples with selective knockdown or knockout of either CRACR2A or CRACR2B

  • Use recombinant protein standards of known concentration for both proteins

  • Create calibration curves with purified proteins to quantify cross-reactivity

• Molecular weight discrimination:

  • Leverage differences in molecular weight between CRACR2A (longer isoform ~95 kDa, shorter isoform ~45 kDa) and CRACR2B

  • Use high-resolution SDS-PAGE with extended run times to separate closely migrating bands

  • Consider using gradient gels (4-20%) for improved resolution

• Immunodepletion approaches:

  • Sequentially deplete lysates of one protein using specific antibodies

  • Analyze the depleted lysate for the second protein

  • This approach can reveal the degree of cross-reactivity between antibodies

• Mass spectrometry validation:

  • Perform immunoprecipitation followed by mass spectrometry analysis

  • Identify protein-specific peptides that distinguish between CRACR2A and CRACR2B

  • Quantify relative abundances of both proteins in experimental samples

These methodological approaches enable researchers to confidently distinguish between CRACR2A and CRACR2B, avoiding misattribution of functions and ensuring accurate interpretation of experimental results in calcium signaling and immunological research.

How might CRACR2B antibodies contribute to understanding immunological disorders beyond known CRACR2A mutations?

CRACR2B antibodies hold significant potential for expanding our understanding of immunological disorders through several research avenues:

• Comparative expression profiling:

  • Analyze CRACR2B expression across patient cohorts with various immunodeficiencies

  • Compare expression patterns with the established CRACR2A-associated immunodeficiency profile

  • Correlate CRACR2B expression levels with specific immune cell defects and clinical phenotypes

• Functional compensation investigation:

  • Examine whether CRACR2B upregulation compensates for CRACR2A deficiency in patients

  • Use CRACR2B antibodies to track potential protein upregulation in patients with CRACR2A mutations

  • Investigate whether artificially increasing CRACR2B can rescue cellular defects in CRACR2A-deficient cells

• Signaling pathway cross-talk:

  • Map CRACR2B involvement in calcium signaling networks beyond the established ORAI1/STIM1 interaction

  • Investigate CRACR2B's potential role in JNK phosphorylation pathways similar to CRACR2A

  • Assess whether CRACR2B participates in distinct signaling cascades affecting immune cell function

• Tissue-specific functions:

  • Compare CRACR2B expression and function across different immune cell populations and tissues

  • Identify cell type-specific binding partners that might contribute to specialized functions

  • Investigate whether CRACR2B has unique roles in specific immune cell subsets

• Genetic screening approaches:

  • Develop antibody-based screening methods to identify patients with abnormal CRACR2B expression

  • Correlate CRACR2B variants with clinical phenotypes in immunodeficiency cohorts

  • Use CRACR2B antibodies to validate functional consequences of newly identified genetic variants

These research directions could potentially reveal CRACR2B as an independent contributor to immune disorders or as a modifier of CRACR2A-associated pathologies, opening new diagnostic and therapeutic possibilities.

What methodological advances might improve CRACR2B antibody sensitivity for detecting low abundance expression?

Enhancing CRACR2B antibody sensitivity for detecting low abundance expression requires innovative methodological approaches:

• Signal amplification technologies:

  • Tyramide signal amplification (TSA) can enhance chromogenic or fluorescent signals up to 100-fold

  • Proximity ligation assay (PLA) variants can detect single molecule interactions

  • Quantum dot-conjugated secondary antibodies provide brighter, more photostable signals

  • Poly-HRP detection systems offer substantially improved sensitivity over conventional HRP methods

• Sample preparation optimization:

  • Protein concentration methods (TCA precipitation, methanol-chloroform extraction)

  • Subcellular fractionation to enrich for compartments with higher CRACR2B concentration

  • Immunoprecipitation followed by Western blotting to concentrate target proteins

  • Optimized fixation protocols that preserve epitope accessibility while maintaining tissue architecture

• Advanced microscopy techniques:

  • Super-resolution microscopy methods (STORM, PALM, STED) to detect sparse protein molecules

  • Light sheet microscopy for improved signal-to-noise in thick tissue sections

  • Lattice light-sheet microscopy for dynamic imaging of low abundance proteins

  • Expansion microscopy to physically magnify samples for improved detection

• Novel antibody engineering approaches:

  • Development of recombinant antibodies with optimized binding kinetics

  • Nanobodies or single-chain variable fragments with improved tissue penetration

  • Bispecific antibodies targeting multiple CRACR2B epitopes simultaneously

  • Antibody fragments optimized for specific applications (e.g., Fab fragments for tissue penetration)

• Computational enhancement:

  • Machine learning algorithms for signal enhancement and background reduction

  • Deconvolution techniques to improve signal resolution in microscopy

  • Automated image analysis workflows optimized for low abundance protein detection

These methodological advances collectively represent a toolkit that researchers can employ to detect and quantify CRACR2B expression even in challenging samples with naturally low abundance or limited specimen availability.