NBEAL2 Antibody, Biotin conjugated

What is NBEAL2 and why is it significant in hematological research?

NBEAL2 (Neurobeachin-Like 2) is a large protein (303 kDa) that plays a critical role in megakaryocyte and platelet biology. It contains BEACH, ARM, Con A-like lectin, PH, and WD40 domains and is essential for alpha-granule biogenesis in platelets. The significance of NBEAL2 in hematological research stems from its causative role in Gray Platelet Syndrome (GPS), a rare bleeding disorder characterized by macrothrombocytopenia and absence of alpha-granules in platelets . Studying NBEAL2 provides insights into fundamental mechanisms of platelet granule formation, secretion, and function in hemostasis, thrombosis, and wound healing processes .

How does NBEAL2 function molecularly in alpha-granule biogenesis?

NBEAL2 functions as a critical mediator of alpha-granule cargo retention during megakaryocyte development and platelet formation. Mechanistically, NBEAL2:

• Colocalizes and coimmunoprecipitates with P-selectin in megakaryocytes, proplatelets, and platelets

• Regulates protein trafficking through RAB5 and RAB7 endosomes to P-selectin-containing alpha-granules

• Prevents alpha-granule cargo proteins from entering RAB11-associated compartments, which would lead to their loss from megakaryocytes

• May interact directly with P-selectin to facilitate cargo retention

Without functional NBEAL2, proteins destined for alpha-granules are misrouted and secreted, resulting in platelets lacking these important granules .

What distinguishes biotin-conjugated NBEAL2 antibodies from other formats?

Biotin-conjugated NBEAL2 antibodies offer distinct advantages compared to other formats:

Biotin-conjugated antibodies are particularly valuable for sensitive detection systems and multiplex applications due to their strong affinity with streptavidin/avidin and versatility in detection methods .

What are the optimal applications for biotin-conjugated NBEAL2 antibodies?

Biotin-conjugated NBEAL2 antibodies are optimally suited for:

• Immunohistochemistry (IHC): The biotin-streptavidin system provides enhanced signal amplification for visualizing NBEAL2 in tissue sections, particularly in bone marrow samples containing megakaryocytes.

• Flow cytometry: Useful for detection of NBEAL2 in platelets and megakaryocytes with streptavidin-conjugated fluorophores.

• Immunoprecipitation: The strong biotin-streptavidin interaction facilitates efficient pull-down of NBEAL2 and associated proteins.

• Chromatin immunoprecipitation (ChIP): For studying GATA1 binding to NBEAL2 regulatory regions, as GATA1 regulates NBEAL2 expression .

• Multiplexed immunoassays: Biotin conjugates enable multi-protein detection systems when combined with other non-biotin conjugated antibodies.

For optimal working dilutions, researchers should perform titration experiments, with recommended starting dilutions of 1:500-1:2000 for most applications .

How should sample preparation be optimized for detection of NBEAL2 in platelets?

Optimizing sample preparation for NBEAL2 detection in platelets requires special consideration due to NBEAL2's large size (303 kDa) and association with alpha-granules:

• Platelet isolation:

  • Use acid-citrate-dextrose (ACD) as anticoagulant

  • Perform gentle centrifugation (180g for 10 minutes) to obtain platelet-rich plasma

  • Wash platelets in buffer containing prostaglandin E1 to prevent activation

• Protein extraction:

  • Use RIPA buffer supplemented with protease inhibitor cocktail

  • Include phosphatase inhibitors if studying phosphorylation states

  • Sonicate briefly (3-5 pulses) to help solubilize membrane-associated NBEAL2

• For immunoblotting:

  • Use low percentage gels (6-8%) or gradient gels to resolve the large 303 kDa protein

  • Transfer at low voltage (30V) overnight at 4°C for efficient transfer of large proteins

  • Use positive controls such as HeLa cell lysates that express NBEAL2

• For microscopy:

  • Fix platelets with 4% paraformaldehyde

  • Perform mild permeabilization with 0.1% Triton X-100

  • Co-stain with P-selectin antibodies to confirm localization to alpha-granules

For NBEAL2 detection in patients with suspected GPS, note that protein levels may be significantly reduced, requiring loading more protein and extending exposure times .

What controls should be included when using biotin-conjugated NBEAL2 antibodies?

A comprehensive control strategy for biotin-conjugated NBEAL2 antibody experiments should include:

Positive controls:

• HeLa cell lysates (confirmed to express NBEAL2)

• Normal human platelets (high NBEAL2 expression)

• Recombinant NBEAL2 protein (if available)

Negative controls:

• Platelets from GPS patients with NBEAL2 mutations (reduced or absent expression)

• Samples from NBEAL2 knockout mice

• Isotype control antibody (rabbit IgG-biotin with same concentration)

Technical controls:

• Blocking with excess unlabeled antibody to demonstrate specificity

• Pre-absorption with immunizing peptide (AA 1360-1574)

• Endogenous biotin blocking (particularly important in tissues with high biotin content)

• Streptavidin-only control (no primary antibody) to assess background

Validation controls:

• Parallel testing with different antibody clones targeting distinct NBEAL2 epitopes

• Correlation with RNA expression data

• Verification using siRNA knockdown experiments

How can biotin-conjugated NBEAL2 antibodies be used to investigate NBEAL2-P-selectin interactions?

Investigating NBEAL2-P-selectin interactions using biotin-conjugated NBEAL2 antibodies can be approached through several advanced methodologies:

• Co-immunoprecipitation with proximity labeling:

  • Use biotin-conjugated NBEAL2 antibody for immunoprecipitation

  • Analyze precipitated complexes for P-selectin by western blotting

  • Alternatively, perform reverse co-IP with P-selectin antibodies

  • Validate interactions using crosslinking approaches before precipitation

• Proximity ligation assay (PLA):

  • Incubate fixed platelets or megakaryocytes with biotin-NBEAL2 antibody and P-selectin antibody

  • Add streptavidin-linked DNA probe and secondary antibody-linked DNA probe

  • If proteins are in close proximity (<40 nm), DNA probes can be ligated and amplified

  • Visualize amplified DNA as fluorescent spots indicating protein-protein interactions

• FRET microscopy:

  • Use biotin-conjugated NBEAL2 antibody with streptavidin-conjugated donor fluorophore

  • Label P-selectin with acceptor fluorophore-conjugated antibody

  • Measure energy transfer as indication of protein proximity

• Super-resolution microscopy:

  • Utilize biotin-conjugated NBEAL2 antibody with streptavidin-conjugated fluorophores

  • Apply STORM or PALM techniques to visualize nanoscale colocalization

  • Quantify colocalization coefficient between NBEAL2 and P-selectin

Previous research has shown that NBEAL2 colocalizes with P-selectin in megakaryocytes, proplatelets, and platelets, and these proteins have been demonstrated to coimmunoprecipitate, suggesting direct interaction or presence in the same complex .

How can biotin-conjugated NBEAL2 antibodies help differentiate between NBEAL2 mutations and GATA1 mutations in Gray Platelet Syndrome?

Biotin-conjugated NBEAL2 antibodies can be instrumental in distinguishing between NBEAL2 and GATA1 mutations in Gray Platelet Syndrome through differential expression analysis:

Methodological approach:

• Western blot analysis:

  • Prepare platelet lysates from patients with Gray Platelet Syndrome

  • Run on SDS-PAGE and transfer to membranes

  • Probe with biotin-conjugated NBEAL2 antibody and streptavidin-HRP

  • Compare expression patterns:

    • NBEAL2 mutations: May show truncated proteins or reduced expression depending on the mutation type

    • GATA1 mutations: Show dramatically reduced or absent NBEAL2 expression

  • Include integrin β3 as loading control

• Flow cytometry:

  • Label fixed and permeabilized platelets with biotin-NBEAL2 antibody

  • Add streptavidin-fluorophore for detection

  • Quantify NBEAL2 expression levels

• Immunofluorescence microscopy:

  • Examine NBEAL2 localization patterns in patient-derived megakaryocytes

  • Co-stain with P-selectin to assess alpha-granule formation

Expected findings:

• Patients with NBEAL2 W2480X mutation: Show markedly decreased NBEAL2 expression

• Patients with GATA1 D218Y mutation: Show absent NBEAL2 expression

• Patients with GATA1 D218G mutation: Show significantly reduced NBEAL2 expression

This distinction is clinically relevant as GATA1 mutations affect multiple hematopoietic lineages beyond platelets, potentially requiring different management strategies.

What approaches can be used to study NBEAL2 trafficking dynamics in live megakaryocytes?

Studying NBEAL2 trafficking dynamics in live megakaryocytes requires sophisticated approaches that can leverage biotin-conjugated antibodies:

• Cell-permeable biotin-conjugated antibody fragments:

  • Generate Fab or scFv fragments of NBEAL2 antibodies

  • Conjugate with biotin and cell-penetrating peptides

  • Introduce into cultured megakaryocytes

  • Add streptavidin-conjugated quantum dots for long-term tracking

• Correlative light-electron microscopy (CLEM):

  • Label live megakaryocytes with biotin-NBEAL2 antibody fragments

  • Perform live-cell imaging to track movements

  • Fix at specific timepoints

  • Process for electron microscopy to resolve subcellular localization

• Multi-color live imaging:

  • Combine biotin-NBEAL2 antibody (with streptavidin-fluorophore) with fluorescently-labeled RAB proteins:

    • RAB5 for early endosomes

    • RAB7 for late endosomes

    • RAB11 for recycling endosomes

  • Track colocalization over time to map NBEAL2 trafficking pathways

• Optogenetic approaches:

  • Use biotin-conjugated NBEAL2 antibodies with streptavidin-photoactivatable proteins

  • Induce protein aggregation or dissociation at specific timepoints

  • Observe effects on alpha-granule formation and cargo retention

Research has shown that NBEAL2 is involved in preventing alpha-granule cargo from entering RAB11-positive compartments, which would lead to their secretion rather than storage in granules . Time-resolved studies could further elucidate how NBEAL2 directs cargo from RAB5/RAB7 compartments to mature alpha-granules.

How can researchers troubleshoot high background when using biotin-conjugated NBEAL2 antibodies?

High background is a common challenge when using biotin-conjugated antibodies. Here's a systematic approach to troubleshoot this issue:

Common causes and solutions:

• Endogenous biotin interference:

  • Problem: Tissues and cells naturally contain biotin that can interact with streptavidin detection systems

  • Solution: Use commercial biotin blocking kits (avidin/biotin blocking systems) prior to antibody incubation

  • Alternative: If blocking is insufficient, consider using a different detection system

• Non-specific binding of the antibody:

  • Problem: Primary antibody binds to proteins other than NBEAL2

  • Solution: Increase blocking time/concentration (use 5% BSA or 10% normal serum)

  • Alternative: Pre-absorb antibody with the immunizing peptide (AA 1360-1574) to confirm specificity

• Excessive antibody concentration:

  • Problem: Too much antibody leads to non-specific binding

  • Solution: Perform antibody titration experiments (start with 1:500-1:2000 dilutions)

  • Alternative: Reduce incubation time to minimize non-specific binding

• Over-detection:

  • Problem: Streptavidin-conjugate concentration too high or development time too long

  • Solution: Dilute streptavidin conjugate (1:1000-1:5000) and carefully monitor development

  • Alternative: Switch to a less sensitive detection system for high-abundance targets

• Tissue/cell autofluorescence (for fluorescent detection):

  • Problem: Natural fluorescence from the sample obscures specific signal

  • Solution: Use autofluorescence quenching reagents

  • Alternative: Select detection fluorophores with emission spectra distinct from autofluorescence

Decision tree for systematic troubleshooting:

• First add appropriate controls (isotype control, secondary-only control)

• If all samples show high background → Focus on blocking and detection system

• If only specific samples show high background → Consider sample-specific factors (fixation, autofluorescence)

• If background persists despite optimization → Consider alternative antibody clones or detection methods

What strategies can optimize detection of low NBEAL2 expression in patient samples with mutations?

Detecting NBEAL2 in samples from patients with mutations presents challenges due to potentially low expression levels. Here are optimized approaches:

• Sample enrichment techniques:

  • Increase protein loading (up to 50-100 μg per lane for Western blots)

  • Immunoprecipitate NBEAL2 prior to detection to concentrate the protein

  • Use density gradient centrifugation to enrich platelets from patient blood

• Signal amplification methods:

  • Employ tyramide signal amplification (TSA) with biotin-conjugated antibodies

  • Use poly-HRP detection systems that provide multiple HRP molecules per binding event

  • Consider QD-streptavidin conjugates which provide brighter, more photostable signals

• Sensitive detection systems:

  • For Western blotting: Use highly sensitive ECL substrates (e.g., femto-level detection)

  • For microscopy: Use high-sensitivity cameras with extended exposure times

  • For flow cytometry: Increase PMT voltage and use narrow bandpass filters

• Specialized protocols for truncated proteins:

  • Use antibodies targeting N-terminal epitopes to detect truncated proteins

  • Run gradient gels (4-12%) to better resolve potential truncated products

  • Consider native gel conditions to maintain protein complexes that may stabilize mutant NBEAL2

• Alternative detection methods:

  • Consider RNA-based detection methods (RT-PCR, RNA-FISH) to confirm expression

  • Use mass spectrometry for targeted peptide detection

  • Employ proximity ligation assays which offer single-molecule sensitivity

Research has shown that even in patients with NBEAL2 mutations (e.g., homozygous splice mutation predicted to result in M1908X), some full-length NBEAL2 protein may still be detectable, alongside truncated forms appearing as lower molecular weight bands .

What are the critical considerations when comparing NBEAL2 expression across different cell types using biotin-conjugated antibodies?

When comparing NBEAL2 expression across different cell types using biotin-conjugated antibodies, researchers should consider several critical factors to ensure valid comparisons:

• Cell type-specific optimization:

  • Fixation protocols: Different cell types may require different fixation methods

    • Platelets: 2% paraformaldehyde, 10 minutes

    • Megakaryocytes: 4% paraformaldehyde, 15 minutes

    • Cell lines: 2-4% paraformaldehyde or methanol fixation based on epitope accessibility

  • Permeabilization: Adjust concentration and time (0.1-0.5% Triton X-100)

  • Blocking conditions: Cell-specific components may require different blocking agents

• Endogenous biotin variations:

  • Problem: Different cell types contain varying amounts of endogenous biotin

  • Solution: Quantify and normalize for endogenous biotin levels

  • Method: Include avidin/biotin blocking steps with titrated concentrations

• Quantitative considerations:

  • Normalization strategy: Use cell type-appropriate housekeeping proteins

    • For platelets: Integrin β3 or GAPDH

    • For megakaryocytes: β-actin or filamin

    • For other cells: Select based on expression stability

  • Quantification method: Use digital image analysis with background subtraction

  • Statistical approach: Apply appropriate statistical tests for cell type-specific variance

• Validation across detection methods:

  • Confirm findings using at least two independent techniques:

    • Flow cytometry for quantitative single-cell analysis

    • Western blotting for molecular weight confirmation

    • Immunofluorescence for localization patterns

    • RT-qPCR for mRNA expression correlation

• Benchmarking with reference samples:

  • Include positive controls with known NBEAL2 expression patterns

  • Use samples from NBEAL2-knockout models as negative controls

  • Consider preparing a standard curve using recombinant NBEAL2 protein

Research has demonstrated significant variations in NBEAL2 expression across developmental stages of megakaryocytes, with expression changing throughout differentiation . Careful standardization is essential when comparing such dynamic expression patterns.

How might biotin-conjugated NBEAL2 antibodies facilitate research into alpha-granule cargo sorting mechanisms?

Biotin-conjugated NBEAL2 antibodies can enable several innovative approaches to investigate alpha-granule cargo sorting mechanisms:

• Proteomic identification of NBEAL2 interaction partners:

  • Use biotin-conjugated NBEAL2 antibodies for immunoprecipitation

  • Identify co-precipitated proteins via mass spectrometry

  • Map the interaction network involved in cargo sorting

  • Compare results from wild-type versus GPS patient samples

• Spatiotemporal mapping of NBEAL2 during granule biogenesis:

  • Utilize super-resolution microscopy with biotin-NBEAL2 antibodies

  • Track NBEAL2 localization relative to cargo proteins during megakaryocyte maturation

  • Correlate with known trafficking markers (RAB5, RAB7, RAB11)

  • Develop 4D models of granule formation and cargo sorting

• In vitro reconstitution assays:

  • Isolate membrane fractions containing NBEAL2 using biotin-conjugated antibodies

  • Reconstitute with fluorescently-labeled cargo proteins

  • Assess cargo retention capacity under various conditions

  • Test effects of mutations or post-translational modifications

• CRISPR-Cas9 domain mapping:

  • Generate domain-specific NBEAL2 mutations (BEACH, ARM, WD40)

  • Use biotin-conjugated antibodies to assess remaining binding interactions

  • Map domain-specific functions in the cargo sorting process

  • Identify minimal functional units required for proper granule formation

Research has established that NBEAL2 prevents alpha-granule cargo from entering RAB11-positive compartments, thus preventing secretion . Further studies using these approaches could elucidate precisely how NBEAL2 recognizes and retains specific cargo proteins during granule biogenesis.

What potential exists for developing therapeutic approaches targeting the NBEAL2 pathway in thrombotic disorders?

The NBEAL2 pathway represents a promising therapeutic target for thrombotic disorders, with several potential approaches:

• Rationale for targeting NBEAL2:

  • NBEAL2-deficient mice show protection from arterial thrombosis and thrombo-inflammatory brain infarction

  • Modulating rather than eliminating alpha-granule function could provide antithrombotic benefits while preserving hemostasis

• Potential therapeutic strategies:

  Approach	Mechanism	Development Considerations	Biotin-NBEAL2 Antibody Application
  Selective NBEAL2 inhibitors	Partial inhibition of alpha-granule formation	Need to maintain some granule function for hemostasis	Target validation and screening assay development
  Cargo-specific modulators	Block retention of prothrombotic factors while preserving others	Requires detailed understanding of cargo-specific interactions	Mapping interaction interfaces
  NBEAL2-P-selectin interaction blockers	Disrupt specific protein-protein interactions	May have more selective effects than global inhibition	Epitope mapping and interaction studies
  Gene therapy for GPS	Restore NBEAL2 function in patients with GPS	Requires megakaryocyte-specific delivery systems	Efficacy assessment in patient cells

• Biomarker development:

  • Use biotin-conjugated NBEAL2 antibodies to develop quantitative assays for NBEAL2 levels

  • Correlate NBEAL2 expression/activity with thrombotic risk

  • Monitor alpha-granule content as predictors of thrombotic events or therapeutic response

• Preclinical model development:

  • Generate conditional or inducible NBEAL2 knockout models

  • Develop NBEAL2 variants with altered cargo specificity

  • Evaluate hemostatic/thrombotic balance in these models

Nbeal2-knockout mice exhibit protection from thrombo-inflammatory brain infarction following focal cerebral ischemia, suggesting that modulation of this pathway could provide therapeutic benefits in stroke and other thrombotic conditions .

How can advanced imaging techniques with biotin-conjugated NBEAL2 antibodies enhance our understanding of platelet granule release dynamics?

Advanced imaging techniques using biotin-conjugated NBEAL2 antibodies can revolutionize our understanding of platelet granule release dynamics:

• Intravital microscopy applications:

  • Develop permeable biotin-NBEAL2 antibody fragments

  • Administer intravenously with streptavidin-conjugated quantum dots

  • Image alpha-granule dynamics during thrombus formation in vivo

  • Correlate granule movements with hemostatic/thrombotic events

• Single-molecule imaging approaches:

  • Utilize biotin-NBEAL2 antibodies with streptavidin-conjugated fluorophores

  • Apply techniques like PALM or STORM for nanoscale resolution

  • Track individual alpha-granules during activation processes

  • Quantify release kinetics with unprecedented precision

• Correlative light-electron microscopy (CLEM):

  • Locate NBEAL2-positive structures via fluorescence microscopy

  • Process the same sample for electron microscopy

  • Generate 3D reconstructions of granule ultrastructure

  • Link molecular composition with structural features

• Lattice light-sheet microscopy:

  • Use biotin-NBEAL2 antibodies to label granules in live platelets

  • Apply lattice light-sheet for rapid 3D imaging with minimal phototoxicity

  • Capture granule dynamics during platelet activation

  • Quantify fusion events and cargo release patterns

• FRET-based release sensors:

  • Develop FRET pairs with biotin-NBEAL2 antibody and cargo proteins

  • Monitor FRET signal changes during granule release

  • Quantify release kinetics with millisecond temporal resolution

  • Correlate with calcium signaling and other activation pathways

These advanced imaging approaches could address fundamental questions about how NBEAL2 participates in granule retention during platelet activation and whether it plays any role in the selective release of granule contents, a phenomenon that has been described but remains poorly understood mechanistically.