nbeal2 Antibody

What is NBEAL2 and why is it significant in research?

NBEAL2 (Neurobeachin-like protein 2) is a large protein approximately 302.5 kDa in size that plays a crucial role in thrombopoiesis and is involved in the development or secretion of alpha-granules in platelets. Mutations in the NBEAL2 gene lead to Grey Platelet Syndrome (GPS), a rare autosomal recessive bleeding disorder characterized by macro-thrombocytopenia and α-granule-deficient platelets . NBEAL2 is significant in research because it has been linked to various immune functions beyond platelet disorders, including T cell regulation and autoimmunity, making it a valuable target for immunological research .

What are the main types of NBEAL2 antibodies available for research?

Several types of NBEAL2 antibodies are available for research applications, including:

• Rabbit monoclonal antibodies (e.g., EPR14501(B))

• Rabbit polyclonal antibodies

• Goat polyclonal antibodies

• Custom-designed antibodies against specific epitopes (e.g., antibodies against exon 1-encoded peptides)

These antibodies come in both conjugated and unconjugated forms and are suitable for various applications including Western blotting, immunofluorescence, immunohistochemistry, and ELISA .

What are the most common applications for NBEAL2 antibodies?

According to the available research data, NBEAL2 antibodies are primarily used for:

• Western blotting (WB) to detect NBEAL2 protein expression

• Immunofluorescence (IF) to determine subcellular localization of NBEAL2

• Immunohistochemistry (IHC) to examine tissue expression patterns

• ELISA for quantitative protein detection

• Co-immunoprecipitation to study protein-protein interactions (particularly important for identifying NBEAL2 interaction partners)

How should I optimize Western blot protocols for detecting NBEAL2?

When optimizing Western blot protocols for NBEAL2 detection:

• Protein Extraction: Consider both soluble and insoluble fractions as NBEAL2 has been detected in both, as demonstrated in Dami cell lysates .

• Gel Selection: Use low percentage gels (3-8% gradient gels) to adequately resolve the large 303 kDa NBEAL2 protein.

• Transfer Conditions: Employ longer transfer times (overnight at low voltage) or semi-dry transfer systems optimized for large proteins.

• Antibody Selection: The EPR14501(B) antibody has been validated at 1/1000 dilution for human platelet lysates .

• Controls: Include both positive controls (human platelet lysates) and negative controls (tissues with low NBEAL2 expression like skeletal muscle) .

• Loading: Load adequate protein (≥10 μg of total protein) to ensure detection of this large, relatively low-abundance protein .

What is the best approach for studying NBEAL2 protein interactions in T cells?

Based on published research methodologies:

• Cell Preparation: Isolate primary T cells and activate them (e.g., with anti-CD3/CD28) to increase NBEAL2 expression .

• Co-Immunoprecipitation: Use either anti-NBEAL2 antibodies or antibodies against suspected interaction partners (e.g., CTLA-4) for pull-down experiments .

• Mass Spectrometry Analysis: For unbiased identification of interaction partners, perform IP followed by mass spectrometry as described in recent studies that identified 74 NBEAL2 interaction partners .

• Validation: Confirm key interactions by reverse co-IP and techniques like proximity ligation assay.

• Controls: Include samples from NBEAL2-deficient individuals/cells as specificity controls for immunoprecipitation experiments .

• Network Analysis: Use STRING database for network analysis of identified partners .

How can I accurately assess NBEAL2 expression across different tissue types?

To accurately assess NBEAL2 expression across tissues:

• Transcript Analysis: Utilize qPCR as demonstrated in studies showing highest expression in CD33+ cells (54.3-fold higher than skeletal muscle), followed by peripheral leukocytes, bone marrow, lung, esophagus, and cervix .

• Protein Detection: Use validated NBEAL2 antibodies for Western blotting and immunohistochemistry to confirm transcript findings at the protein level .

• Subcellular Localization: Employ immunofluorescence with specific NBEAL2 antibodies to determine subcellular localization (preliminary findings suggest cytoplasmic localization) .

• Controls: Include tissues known to have high (CD33+ cells, platelets) and low (skeletal muscle, brain) NBEAL2 expression .

• Cross-Species Validation: Consider using conserved epitope antibodies that recognize both human and mouse NBEAL2 for comparative studies .

How can NBEAL2 antibodies be used to study T cell regulation in autoimmune disorders?

NBEAL2 antibodies can be instrumental in studying T cell dysregulation in autoimmunity through several approaches:

• CTLA-4 Expression Analysis: Use flow cytometry with NBEAL2 and CTLA-4 antibodies to assess CTLA-4 expression in different T cell subsets from patients with NBEAL2 mutations or from NBEAL2 knockdown models .

• Differential Analysis of T Cell Subsets: Compare CTLA-4 expression in effector T cells versus regulatory T cells, as research shows NBEAL2 deficiency specifically impacts CTLA-4 expression in effector T cells but not Tregs .

• Trafficking Studies: Employ confocal microscopy with NBEAL2 and CTLA-4 antibodies to track CTLA-4 trafficking and recycling in T cells .

• Functional Assays: Perform suppression assays to assess how NBEAL2 deficiency impacts T cell function and immune regulation .

• Therapeutic Response Monitoring: Use NBEAL2 antibodies to monitor patients with GPS who receive CTLA-4-immunoglobulin therapy, as suggested by recent studies .

What experimental approaches can differentiate the roles of NBEAL2 in platelets versus immune cells?

To differentiate NBEAL2's roles across different cell types:

• Cell-Specific Knockout Models: Generate conditional Nbeal2 knockout mice with cell-specific deletions (platelets vs. T cells vs. other immune cells) .

• Bone Marrow Chimeras: Create chimeric mice through bone marrow transplantation experiments to distinguish hematopoietic vs. non-hematopoietic functions of NBEAL2 .

• Co-IP Comparative Analysis: Use NBEAL2 antibodies for co-immunoprecipitation in different cell types to identify cell-specific interaction partners .

• Functional Assays:

  • For platelets: Assess aggregation, adhesion under flow, and pro-coagulant activity

  • For T cells: Examine CTLA-4 expression and T cell suppressive function

  • For other immune cells: Analyze granule content and degranulation in neutrophils and NK cells

• Disease Models:

  • Hypoxia-induced pulmonary hypertension for platelet function

  • Autoimmune/inflammatory models for immune cell function

How should researchers interpret conflicting data between NBEAL2 expression and protein function across different experimental systems?

When faced with conflicting data regarding NBEAL2:

• Cell Type Considerations: Recognize that NBEAL2 may have distinct roles in different cell types. For example, it regulates alpha-granule formation in platelets but CTLA-4 expression in T cells .

• Species Differences: While human and mouse NBEAL2 are highly conserved, subtle differences may exist. Consider using antibodies against conserved epitopes (e.g., the 14 amino acid peptide SLEPRRPEEAGAEVC in exon 1) when comparing across species .

• Technical Variables:

  • Antibody specificity: Verify antibody specificity using NBEAL2-deficient controls

  • Protein size: Ensure proper detection of this large 303 kDa protein by using appropriate gel systems and transfer methods

  • Cellular fractionation: Check both soluble and insoluble fractions, as NBEAL2 has been detected in both

• Context-Dependent Functions: NBEAL2's function may depend on its binding partners, which vary by cell type and activation state. Use co-IP followed by mass spectrometry to identify relevant interactors in your specific experimental system .

• Temporal Considerations: Some NBEAL2-related phenotypes develop over time, as seen in the hypoxia-induced pulmonary hypertension model where Nbeal2^−/− mice showed attenuated disease at day 21 but not at day 35 .

What are the most common challenges when working with NBEAL2 antibodies and how can they be addressed?

Common challenges and solutions include:

• Detection of Large Protein: NBEAL2 is approximately 303 kDa, which can be difficult to resolve and transfer.

  • Solution: Use low percentage gels (3-8%), extended transfer times, and specialized transfer systems for large proteins .

• Low Signal Intensity: NBEAL2 expression varies widely across tissues.

  • Solution: Use tissues with known high expression (CD33+ cells, platelets) as positive controls and concentrate samples when working with tissues with lower expression .

• Nonspecific Binding: Large proteins often show cross-reactivity.

  • Solution: Include NBEAL2-deficient samples as negative controls and validate antibodies using multiple techniques (WB, IF, IP) .

• Variability in Expression: NBEAL2 expression can be influenced by cell activation state.

  • Solution: Standardize cell activation protocols, particularly when working with immune cells .

• Subcellular Localization Discrepancies: Conflicting reports on NBEAL2 localization.

  • Solution: Use multiple antibodies targeting different epitopes and confirm with subcellular fractionation approaches .

How should researchers design controls for NBEAL2 antibody validation?

A comprehensive validation approach should include:

• Positive Controls:

  • Cell/tissue types with known high expression: CD33+ cells, peripheral leukocytes, bone marrow, platelets

  • Recombinant NBEAL2 protein (full-length or fragments)

• Negative Controls:

  • NBEAL2-deficient samples (from GPS patients or Nbeal2^−/− mice)

  • Tissues with very low expression (e.g., skeletal muscle)

  • Secondary antibody-only controls

• Specificity Controls:

  • Peptide competition assays using the immunizing peptide

  • Multiple antibodies targeting different epitopes

  • siRNA or CRISPR/Cas9 knockdown of NBEAL2 in relevant cell lines

• Cross-Reactivity Assessment:

  • Testing across species if using for comparative studies

  • Testing related BEACH domain proteins (e.g., LRBA) to ensure specificity

What are the appropriate experimental conditions for using NBEAL2 antibodies in various applications?

Optimal conditions vary by application:

• Western Blotting:

  • Dilution: 1/1000 for anti-NBEAL2 antibody [EPR14501(B)]

  • Sample: 10 μg or more of total protein from relevant tissues

  • Detection: Anti-Rabbit IgG (HRP) secondary antibody at 1/1000 dilution

• Immunoprecipitation:

  • Sample preparation: Lyse cells in appropriate buffer (used for isolating 74 NBEAL2 interaction partners)

  • Antibody amount: Optimize based on specific antibody and application

  • Controls: Include IgG control and NBEAL2-deficient samples

• Immunofluorescence:

  • Fixation: Test both paraformaldehyde and methanol fixation

  • Antibody dilution: Start with manufacturer's recommendation and optimize

  • Co-staining: Consider co-staining with markers of subcellular compartments to determine localization

• Flow Cytometry:

  • Sample: Activated T cells show higher NBEAL2 expression

  • Controls: Include fluorescence minus one (FMO) controls

  • Analysis: When studying CTLA-4, distinguish between surface and total CTLA-4 expression

How might NBEAL2 antibodies contribute to developing therapeutic strategies for Grey Platelet Syndrome?

NBEAL2 antibodies could advance therapeutic development in several ways:

• Biomarker Development: NBEAL2 antibodies could help monitor disease progression and treatment response in GPS patients .

• Mechanism-Based Therapeutics: Studies using NBEAL2 antibodies have revealed CTLA-4 dysregulation in GPS patients with autoimmunity, suggesting CTLA-4-immunoglobulin therapy as a potential treatment approach .

• Screening Platforms: Development of screening assays using NBEAL2 antibodies to identify compounds that can rescue NBEAL2 function or compensate for its absence.

• Targeted Drug Delivery: Understanding NBEAL2's interaction network through co-IP with NBEAL2 antibodies could identify potential drug targets within the same pathway .

• Personalized Medicine Approaches: NBEAL2 antibodies could help classify GPS patients based on protein expression levels and interaction profiles, potentially guiding personalized treatment strategies.

What novel techniques could enhance the specificity and utility of NBEAL2 antibodies in complex experimental systems?

Emerging approaches include:

• Nanobody Development: Engineering smaller antibody fragments that may access epitopes unavailable to conventional antibodies in this large, complex protein.

• Proximity Labeling: Combining NBEAL2 antibodies with proximity labeling techniques (BioID, APEX) to identify transient or weak interaction partners in living cells.

• Super-Resolution Microscopy: Using highly specific NBEAL2 antibodies with techniques like STORM or PALM to precisely locate NBEAL2 within subcellular compartments.

• Multiplexed Imaging: Developing antibody panels for simultaneous detection of NBEAL2 and its interaction partners in tissues and cells .

• Recombinant Antibody Engineering: Creating recombinant antibodies with enhanced specificity for different NBEAL2 domains to distinguish its various functions.

• Antibody-Drug Conjugates: For research purposes, creating NBEAL2 antibody conjugates to deliver compounds that modulate protein function in specific cell types.

How can researchers integrate NBEAL2 antibody data with other omics approaches to gain comprehensive insights into disease mechanisms?

Integration strategies include:

• Multi-Omics Integration:

  • Combine NBEAL2 antibody-based proteomics with transcriptomics to correlate protein expression with mRNA levels across tissues

  • Integrate interactome data from NBEAL2 co-IP studies with phosphoproteomics to identify signaling pathways affected by NBEAL2 deficiency

• Single-Cell Analysis:

  • Use NBEAL2 antibodies for single-cell proteomics or CyTOF to examine cell-specific expression patterns

  • Correlate with single-cell transcriptomics to identify cell populations most affected by NBEAL2 mutations

• Systems Biology Approaches:

  • Network analysis of NBEAL2 interaction partners identified through co-IP and mass spectrometry

  • Pathway enrichment analysis to identify biological processes affected by NBEAL2 deficiency

• Functional Genomics Integration:

  • Combine CRISPR screens with NBEAL2 antibody-based phenotyping to identify genetic modifiers of NBEAL2 function

  • Correlate genetic variants in NBEAL2 with protein expression and localization patterns

• Longitudinal Studies:

  • Use NBEAL2 antibodies to track protein expression and interactions over disease progression, particularly in models like the hypoxia-induced pulmonary hypertension where effects change over time

Table 1: NBEAL2 Expression Across Human Tissues

Table 2: Validated Applications for Common NBEAL2 Antibodies