FCER1A Human 201 a.a

What is FCER1A and what is its biological function?

FCER1A is the alpha subunit of the high-affinity immunoglobulin E (IgE) receptor, primarily expressed on mast cells and basophils. It binds to the Fc region of IgE antibodies and plays a crucial role in initiating allergic responses. The receptor consists of an alpha subunit (FCER1A), a beta subunit, and two gamma subunits, forming a tetrameric complex. FCER1A is responsible for starting the allergic cascade when allergens crosslink IgE antibodies bound to the receptor, leading to cell activation and the release of inflammatory mediators such as histamine, cytokines, and leukotrienes, which contribute to the clinical manifestations of allergic reactions .

What is the structure and composition of the recombinant FCER1A Human 201 a.a. protein?

The recombinant FCER1A Human 201 a.a. protein is a non-glycosylated polypeptide chain containing 201 amino acids produced in E. coli. It is fused to a 6 amino acid His-tag at the C-terminus and purified using proprietary chromatographic techniques. The physical appearance is a sterile filtered colorless solution, formulated in 1x PBS with 50mM Arginine and 0.05% NaN₃. The protein has a purity greater than 90.0% as determined by SDS-PAGE analysis .

How should FCER1A Human 201 a.a. be stored and handled for research applications?

For optimal stability and integrity, FCER1A Human 201 a.a. recombinant protein should be stored below -18°C. While the protein is stable at 4°C for up to one week, long-term storage requires freezing. For extended storage periods, it is recommended to add a carrier protein (0.1% HSA or BSA) to enhance stability. It is crucial to prevent freeze-thaw cycles as they can compromise protein integrity and functionality. When working with the protein, maintain sterile conditions and handle the solution according to standard laboratory practices for recombinant proteins .

What are the known disease associations with FCER1A?

FCER1A has been implicated in several disease conditions, most notably allergic disorders. Among the diseases associated with FCER1A are mast-cell leukemia and allergic asthma. Research has identified FCER1A as a key allergy gene, with certain variations of the gene decisively influencing the production of immunoglobulin E (IgE) antibodies. Additionally, FCER1A dysregulation has been linked to chronic spontaneous urticaria, chronic urticaria, and other immune-mediated inflammatory conditions such as atopic dermatitis. Understanding these disease associations provides important insights for research focused on allergic and inflammatory disease mechanisms .

How can I design experiments to study FCER1A-IgE interactions using the recombinant protein?

To study FCER1A-IgE interactions using recombinant FCER1A Human 201 a.a., consider implementing the following methodological approach:

• Binding Assays: Utilize surface plasmon resonance (SPR) or bio-layer interferometry (BLI) to quantify binding kinetics. Immobilize either the His-tagged FCER1A on a Ni-NTA sensor chip or biotinylated IgE on a streptavidin surface, then measure association and dissociation rates.

• Functional Assays: Establish cell-based systems using mast cell or basophil lines transfected with the complete receptor complex (alpha, beta, and gamma subunits). Then, assess activation using the recombinant FCER1A in competition assays.

• Structural Studies: Employ X-ray crystallography or cryo-electron microscopy to analyze the structural details of the FCER1A-IgE interface, requiring high-purity recombinant protein.

• Crosslinking Experiments: Design assays where the recombinant protein competes with native receptors for IgE binding, and measure downstream signaling events such as calcium flux or mediator release .

What are the optimal conditions for reconstitution and use of FCER1A Human 201 a.a. in cell-based assays?

For optimal reconstitution and use of FCER1A Human 201 a.a. in cell-based assays, follow these methodological guidelines:

• Reconstitution: Reconstitute the lyophilized protein in sterile water or PBS buffer to a concentration of 0.1-1.0 mg/mL, depending on your experimental requirements. Gentle mixing is recommended rather than vigorous vortexing to prevent protein denaturation.

• Buffer Optimization: For cell-based assays, ensure the final buffer composition is compatible with cell viability (pH 7.2-7.4, physiological salt concentration). The formulation containing 1x PBS, 50mM Arginine and 0.05% NaN₃ should be considered when designing the assay, as NaN₃ can be toxic to cells at higher concentrations.

• Concentration Determination: Perform a titration series (typically 0.1-100 nM) to determine the optimal working concentration for your specific assay.

• Control Experiments: Include appropriate positive and negative controls, such as known FCER1A ligands and non-specific proteins of similar size and structure.

• Incubation Conditions: For most mammalian cell-based assays, incubation at 37°C with 5% CO₂ in serum-free or low-serum (0.5-2%) media for 1-4 hours prior to assessing cellular responses is recommended .

How can I validate the functional activity of FCER1A Human 201 a.a. in experimental systems?

To validate the functional activity of FCER1A Human 201 a.a. in experimental systems, implement the following comprehensive methodological approach:

• Binding Validation: Perform an ELISA-based assay where the recombinant FCER1A is immobilized on a plate and its ability to bind purified human IgE is quantified. A dose-response curve with known concentrations of IgE will confirm binding functionality.

• Competition Assays: Utilize flow cytometry to measure the capacity of soluble FCER1A 201 a.a. to compete with cell-surface FCER1A for IgE binding on mast cells or basophils.

• Signaling Inhibition: In cellular systems expressing the complete high-affinity IgE receptor, determine if pre-incubation with recombinant FCER1A 201 a.a. can inhibit IgE-mediated signaling events, such as calcium mobilization or phosphorylation of downstream effectors.

• Mediator Release Assays: Assess whether the recombinant protein affects degranulation and inflammatory mediator release in mast cells or basophils challenged with allergen-IgE complexes.

• Western Blot Analysis: Confirm protein integrity and recognition by specific antibodies against FCER1A using immunoblotting techniques before functional assays .

How can FCER1A Human 201 a.a. be used to study receptor signaling pathways in allergic responses?

FCER1A Human 201 a.a. can be utilized in several sophisticated approaches to elucidate receptor signaling pathways in allergic responses:

• Phosphoproteomic Analysis: Use the recombinant protein as either an antagonist or competitive inhibitor in cellular systems, followed by mass spectrometry-based phosphoproteomics to map signaling networks activated during allergic responses. Compare phosphorylation patterns in the presence versus absence of FCER1A 201 a.a. to identify key regulatory nodes.

• Proximity Labeling: Employ biotinylated FCER1A 201 a.a. in BioID or APEX2 proximity labeling techniques to identify protein interaction networks surrounding the receptor in different cellular contexts.

• Single-Cell Analysis: Combine recombinant FCER1A treatments with single-cell RNA sequencing or CyTOF analysis to characterize heterogeneity in cellular responses across diverse immune cell populations.

• CRISPR Screens: Use FCER1A 201 a.a. as a molecular probe in genome-wide CRISPR screens to identify genes that modulate receptor-mediated signaling pathways.

• Optogenetic Approaches: Develop optogenetically controllable FCER1A variants based on the recombinant protein structure to precisely control and study temporal aspects of receptor activation and downstream signaling cascades .

What approaches can be used to study FCER1A polymorphisms and their impact on receptor function?

To investigate FCER1A polymorphisms and their functional impacts, researchers can implement these advanced methodological approaches:

• Site-Directed Mutagenesis: Generate recombinant FCER1A 201 a.a. variants containing specific polymorphisms identified in genome-wide association studies (GWAS) through site-directed mutagenesis. Compare binding kinetics, thermal stability, and functional properties between wild-type and mutant proteins.

• Structure-Function Analysis: Employ computational modeling and molecular dynamics simulations to predict how specific polymorphisms might alter receptor structure and IgE binding properties, then validate these predictions experimentally.

• Patient-Derived Samples: Isolate primary cells from individuals with different FCER1A genotypes and assess receptor expression, IgE binding capacity, and downstream signaling responses.

• Genome Editing: Use CRISPR-Cas9 to introduce specific FCER1A polymorphisms into relevant cell lines or primary cells, then characterize functional consequences using multiparameter flow cytometry, calcium signaling assays, or mediator release measurements.

• Reporter Systems: Develop cell-based reporter systems expressing FCER1A variants linked to fluorescent or luminescent reporters to quantitatively assess polymorphism effects on receptor expression and function in real-time .

How can FCER1A Human 201 a.a. be utilized in drug discovery for allergic disorders?

FCER1A Human 201 a.a. can be strategically incorporated into drug discovery pipelines for allergic disorders through the following methodological approaches:

• High-Throughput Screening: Develop fluorescence-based or FRET-based assays using labeled FCER1A 201 a.a. to screen compound libraries for molecules that disrupt IgE-FCER1A interactions. The recombinant protein provides a stable, consistent target for primary screening campaigns.

• Fragment-Based Drug Design: Utilize the recombinant protein in NMR-based fragment screening or X-ray crystallography to identify small molecule binding sites that could be exploited for rational drug design.

• Peptide Mimetics: Design peptide mimetics based on the IgE-binding interface of FCER1A, using the recombinant protein to validate binding and inhibitory properties.

• Biologics Development: Engineer antibodies or other biologics that specifically target FCER1A, using the recombinant protein for initial screening, affinity maturation, and in vitro characterization before cellular testing.

• Mechanism of Action Studies: For candidate compounds that modulate allergic responses, determine if they interact directly with FCER1A using biophysical techniques such as isothermal titration calorimetry (ITC), microscale thermophoresis (MST), or surface plasmon resonance (SPR) with the recombinant protein .

How does FCER1A Human 201 a.a. compare structurally and functionally with FCER1A from other species?

A comprehensive comparative analysis of FCER1A Human 201 a.a. with orthologs from other species reveals important evolutionary and functional insights:

What is the relationship between FCER1A and dendritic cell differentiation in immune responses?

The relationship between FCER1A and dendritic cell (DC) differentiation represents a complex aspect of immune system regulation:

• Expression Patterns: While FCER1A is classically associated with mast cells and basophils, research has revealed its presence on specific dendritic cell subsets, particularly plasmacytoid DCs (pDCs). Single-cell transcriptome analyses have identified FCER1A expression as part of the transcriptional signature distinguishing certain DC populations.

• Differentiation Marker: FCER1A expression changes during DC differentiation from CD34+ precursors, with studies identifying it as a marker in DC development pathways. The expression pattern provides insights into the developmental trajectory of distinct DC subsets.

• Functional Implications: In DCs, FCER1A can:

  • Mediate IgE-dependent antigen presentation to T cells

  • Influence cytokine production profiles

  • Modulate DC maturation and migration

  • Contribute to allergic inflammation through altered DC function

• Experimental Approaches: To study this relationship, researchers can:

  • Use flow cytometry to track FCER1A expression during DC differentiation

  • Employ the recombinant protein to modulate DC function in vitro

  • Analyze transcriptional networks coordinating FCER1A expression with DC lineage commitment

• Therapeutic Relevance: Understanding FCER1A in the context of DC biology has implications for developing immunomodulatory strategies targeting allergic disorders through DC-directed approaches .

How can FCER1A Human 201 a.a. be integrated into systems biology approaches to study allergic disorders?

FCER1A Human 201 a.a. can be strategically integrated into systems biology frameworks through these advanced methodological approaches:

• Multi-omics Integration: Utilize the recombinant protein in perturbation experiments followed by integrated analysis of:

  • Transcriptomics (RNA-seq) to identify gene expression changes

  • Proteomics to capture translation-level alterations

  • Metabolomics to assess downstream metabolic consequences

  • Epigenomics to evaluate chromatin remodeling responses
    This multi-layered data can reveal emergent properties of allergic response networks not visible at any single analytical level.

• Network Modeling: Develop computational models incorporating:

  • Protein-protein interaction networks with FCER1A as a central node

  • Signaling pathway models parameterized with binding kinetics data from recombinant protein studies

  • Gene regulatory networks governing cellular responses to FCER1A activation

• Single-Cell Resolution Approaches: Apply the recombinant protein in single-cell experimental systems to:

  • Map cellular heterogeneity in response to FCER1A stimulation or inhibition

  • Identify rare cell populations with unique response patterns

  • Track dynamic changes in cell state during allergic responses

• In Silico Drug Screening: Use structural data and binding characteristics of FCER1A 201 a.a. to:

  • Generate pharmacophore models for virtual screening

  • Predict off-target effects of potential therapeutics

  • Rank candidate molecules for experimental validation

• Clinical Data Integration: Correlate in vitro findings using the recombinant protein with patient-derived data to identify biomarkers and therapeutic targets with translational potential .

What are the common challenges in working with FCER1A Human 201 a.a. and how can they be addressed?

Researchers frequently encounter several challenges when working with FCER1A Human 201 a.a. These methodological issues and their solutions include:

• Protein Stability Issues:

  • Challenge: The recombinant protein may lose activity during storage or experimental handling.

  • Solution: Store at -18°C or below, add carrier proteins (0.1% HSA or BSA) for long-term storage, aliquot to avoid freeze-thaw cycles, and validate activity before critical experiments using binding assays.

• Non-Glycosylation Limitations:

  • Challenge: The E. coli-produced 201 a.a. version lacks glycosylation present in native FCER1A, potentially affecting function.

  • Solution: For glycosylation-dependent studies, compare with the HEK293-produced version (186 a.a.), which contains mammalian glycosylation patterns. Validate findings across both protein variants.

• Buffer Compatibility:

  • Challenge: The storage buffer containing NaN₃ may interfere with certain assays or cell viability.

  • Solution: Consider buffer exchange using desalting columns or dialysis into a compatible buffer system before cellular experiments.

• Aggregation Problems:

  • Challenge: Recombinant FCER1A may form aggregates affecting functional studies.

  • Solution: Centrifuge at 10,000g for 10 minutes before use, filter through a 0.22μm filter if necessary, and confirm monodispersity using dynamic light scattering.

• His-tag Interference:

  • Challenge: The C-terminal His-tag may interfere with certain interactions or assays.

  • Solution: Where critical, compare results with tag-cleaved versions of the protein or validate with antibodies recognizing different epitopes .

How can I optimize immunoassays and binding studies using FCER1A Human 201 a.a.?

To optimize immunoassays and binding studies using FCER1A Human 201 a.a., implement these methodological refinements:

• ELISA Optimization:

  • Coating Conditions: Test different coating buffers (carbonate buffer pH 9.6, PBS pH 7.4) and concentrations (0.1-10 μg/mL) of FCER1A.

  • Blocking Agents: Compare BSA, casein, and commercial blocking buffers for lowest background.

  • Detection System: For maximal sensitivity, use biotinylated detection antibodies or IgE followed by streptavidin-HRP rather than direct HRP conjugates.

  • Calibration Curve: Develop a standard curve using purified IgE (0.1-100 ng/mL) to ensure the assay operates in the linear range.

• Surface Plasmon Resonance Refinements:

  • Surface Chemistry: For His-tagged FCER1A, NTA-functionalized chips provide oriented immobilization, while amine coupling may cause heterogeneous surfaces.

  • Flow Rate Optimization: Use higher flow rates (30-50 μL/min) during association to minimize mass transport limitations.

  • Regeneration Conditions: Develop mild regeneration conditions (10mM glycine pH 2.0-2.5 for 30 seconds) that remove bound analyte without denaturing the immobilized FCER1A.

• Flow Cytometry Applications:

  • Labeling Strategy: Directly label FCER1A with fluorophores at a molar ratio of 2-4 fluorophore molecules per protein for optimal signal-to-noise.

  • Controls: Include unstained cells, isotype controls, and competition with unlabeled protein to confirm specificity.

  • Multiparameter Analysis: Combine with markers for cell activation status to correlate binding with functional outcomes.

• Functional Readouts:

  • Calcium Flux: Load cells with Fluo-4 AM and measure real-time calcium responses to FCER1A-mediated stimulation.

  • Phospho-Flow: Use phospho-specific antibodies to quantify activation of signaling molecules downstream of FCER1A engagement .

What quality control measures should be implemented when working with FCER1A Human 201 a.a.?

Implementation of rigorous quality control measures is essential when working with FCER1A Human 201 a.a. to ensure experimental reproducibility and validity:

• Protein Validation:

  • Purity Assessment: Confirm >90% purity using SDS-PAGE with Coomassie staining before experiments.

  • Identity Confirmation: Verify protein identity via Western blot with anti-FCER1A antibodies and/or mass spectrometry.

  • Endotoxin Testing: As an E. coli-derived product, test for endotoxin contamination using LAL assay, especially for cell-based experiments.

  • Batch Consistency: Compare new batches with reference standards using analytical techniques like SEC-HPLC.

• Functional Qualification:

  • Binding Activity: Establish a standard IgE binding assay (ELISA or SPR) with acceptance criteria for each new lot.

  • Dose-Response Characterization: Determine EC50 values in standard assays to ensure lot-to-lot consistency.

  • Thermal Stability: Perform differential scanning fluorimetry to assess protein stability and compare between batches.

• Storage Monitoring:

  • Stability Program: Implement a regular testing schedule (0, 1, 3, 6 months) to monitor activity retention.

  • Freeze-Thaw Testing: Quantify activity loss after defined numbers of freeze-thaw cycles.

  • Temperature Excursion Studies: Assess impact of temporary storage at suboptimal temperatures.

• Documentation Practices:

  • Certificate of Analysis: Maintain comprehensive documentation including expression system, purification method, buffer composition, concentration, and QC results.

  • Usage Tracking: Implement sample tracking with freeze-thaw counts and experimental outcomes.

  • Standardized Protocols: Develop and validate standard operating procedures for handling and experimental use .

What are the latest research findings regarding FCER1A's role in allergic and inflammatory disorders?

Recent research has expanded our understanding of FCER1A's role in allergic and inflammatory disorders, revealing several groundbreaking insights:

• Genetic Associations: GWAS studies have firmly established FCER1A as a key allergy gene, with specific variations decisively influencing IgE antibody production. The gene encodes the alpha chain of the high-affinity IgE receptor, which plays a major role in controlling allergic responses. These genetic findings provide a molecular basis for allergen susceptibility and potential personalized medicine approaches .

• Beyond Classical Allergies: Emerging evidence implicates FCER1A in a broader range of inflammatory conditions beyond classical allergic disorders, including chronic spontaneous urticaria and certain autoimmune conditions. This suggests a more versatile role in immune regulation than previously recognized .

• Novel Cell Populations: While traditionally associated with mast cells and basophils, recent single-cell transcriptomic analyses have identified FCER1A expression in specific dendritic cell populations, suggesting its involvement in antigen presentation and T cell priming during allergic responses. This expands our understanding of how allergic responses are initiated and propagated .

• Regulatory Mechanisms: New studies have uncovered previously unrecognized regulatory mechanisms controlling FCER1A expression and signaling, including epigenetic modifications, non-coding RNAs, and posttranslational modifications that fine-tune receptor function in different microenvironments.

• Tissue-Specific Roles: Research has demonstrated tissue-specific functions of FCER1A, with distinct roles in barrier tissues like skin and respiratory mucosa compared to its functions in circulating immune cells, providing insights into localized allergic responses .

How might FCER1A Human 201 a.a. be used in developing novel therapeutic approaches for allergic diseases?

FCER1A Human 201 a.a. represents a valuable tool for developing innovative therapeutic approaches for allergic diseases through several methodological strategies:

• Competitive Antagonism: The recombinant protein can be engineered to create high-affinity decoy receptors that sequester IgE without triggering cellular activation. These modified versions could be tested as competitive antagonists to prevent allergen-IgE-FCER1A interactions in vivo.

• Epitope Mapping: Using the recombinant protein in systematic binding studies can identify critical binding epitopes for structure-based drug design. This approach enables the development of small molecule inhibitors or peptide mimetics that specifically disrupt IgE-FCER1A interactions.

• Antibody Development: FCER1A 201 a.a. serves as an ideal antigen for generating and screening monoclonal antibodies that block IgE binding without triggering receptor activation. These antibodies could be humanized and developed as therapeutic biologics.

• Nanoparticle-Based Approaches: The recombinant protein can be conjugated to nanoparticles to create multivalent inhibitors with enhanced avidity and improved pharmacokinetics compared to soluble proteins.

• Combination Therapies: In vitro systems using FCER1A 201 a.a. can facilitate the screening of combination therapies targeting multiple aspects of the allergic cascade simultaneously, potentially leading to synergistic therapeutic effects.

• Personalized Medicine Applications: The recombinant protein can be used in functional assays with patient-derived samples to predict individual responsiveness to FCER1A-targeted therapies, enabling a precision medicine approach to allergic disease treatment .

What emerging technologies could enhance the study of FCER1A structure, function, and interactions?

Cutting-edge technologies are revolutionizing our ability to investigate FCER1A structure, function, and interactions at unprecedented resolution:

• Cryo-Electron Microscopy: Recent advances in cryo-EM now enable visualization of membrane receptor complexes at near-atomic resolution. Applied to FCER1A, this technology could reveal dynamic conformational changes during IgE binding and receptor activation, particularly when combined with the recombinant 201 a.a. protein in reconstituted membrane systems.

• AlphaFold2 and Computational Approaches: AI-powered protein structure prediction tools can now generate highly accurate models of protein-protein interactions. These computational approaches can predict how FCER1A interfaces with IgE and other binding partners, guiding experimental design and drug discovery efforts.

• CRISPR-Based Screening Platforms: High-throughput CRISPR screens combined with single-cell readouts can systematically identify genes that modulate FCER1A expression, trafficking, and signaling in relevant cell types, revealing new regulatory mechanisms and therapeutic targets.

• Spatial Transcriptomics and Proteomics: These technologies enable mapping of FCER1A expression and activity within intact tissues, preserving spatial context and cellular neighborhoods to understand receptor function in complex physiological environments.

• Organoid and Microphysiological Systems: Advanced 3D culture systems incorporating multiple cell types can recapitulate tissue-level allergic responses, allowing study of FCER1A function in more physiologically relevant contexts than traditional 2D cultures.

• Live-Cell Biosensors: Genetically encoded biosensors can track FCER1A activation and downstream signaling events in real-time within living cells, providing dynamic information about receptor function not captured by endpoint assays .