NBEAL2 Antibody, FITC conjugated

What is NBEAL2 and why is it important in research?

NBEAL2 (Neurobeachin-like protein 2) is a member of the BEACH domain protein family that plays crucial roles in vesicle transport, fusion of vesicles with cell membranes, and granule formation. It is particularly important in hematopoietic cells, where it regulates the development and secretion of alpha-granules in platelets, influences mast cell homeostasis, and affects neutrophil function . Loss-of-function mutations in NBEAL2 cause Gray Platelet Syndrome (GPS), a rare bleeding disorder characterized by thrombocytopenia, enlarged platelets lacking alpha-granules, and variable bleeding tendencies .

What specific applications are NBEAL2 antibodies suitable for?

NBEAL2 antibodies, including FITC-conjugated versions, are primarily used for immunological applications such as ELISA, immunohistochemistry on paraffin-embedded tissues (IHC-P), and immunocytochemistry/immunofluorescence (ICC/IF) . These antibodies enable researchers to detect and visualize NBEAL2 protein expression in various tissues and cell lines, particularly in hematopoietic lineages where NBEAL2 function is critical.

How should NBEAL2 antibodies be stored and handled to maintain optimal activity?

NBEAL2 antibodies, including FITC-conjugated versions, should be stored at -20°C or -80°C upon receipt to maintain activity . Repeated freeze-thaw cycles should be avoided as they can degrade antibody quality and fluorescence intensity. The antibodies are typically supplied in a buffer containing preservatives (such as 0.03% Proclin 300) and stabilizers (such as 50% Glycerol, 0.01M PBS, pH 7.4) . When working with FITC-conjugated antibodies, it's important to protect them from light exposure to prevent photobleaching of the fluorophore.

What controls should be included when using NBEAL2 antibodies in immunofluorescence studies?

For immunofluorescence studies using FITC-conjugated NBEAL2 antibodies, researchers should include:

• Positive control: Tissues or cell lines known to express NBEAL2, such as HepG2 cells or hematopoietic lineages

• Negative control: Tissues from NBEAL2-knockout models (Nbeal2^-/-) or cells with CRISPR/Cas9-mediated NBEAL2 deletion

• Isotype control: FITC-conjugated IgG isotype control at the same concentration as the NBEAL2 antibody to assess non-specific binding

• Secondary antibody control: When using indirect immunofluorescence methods, include a sample with secondary antibody only (e.g., Alexa-Fluor 488-conjugated Goat Anti-Rabbit IgG)

These controls help distinguish specific NBEAL2 staining from background fluorescence or non-specific binding.

What concentration of NBEAL2 antibody is optimal for different applications?

Based on the search results, the following concentrations are recommended:

• For ICC/IF: 1/100 dilution has been verified with human cell lines such as HepG2

• For IHC-P: 1/100 dilution has been shown effective with human tissues including ovarian cancer samples

• For ELISA: Specific dilutions should be optimized, as the polyclonal antibody is suitable for this application

Researchers should perform titration experiments to determine optimal concentrations for their specific samples and experimental conditions.

How can NBEAL2 antibodies be used to investigate Gray Platelet Syndrome pathophysiology?

NBEAL2 antibodies are valuable tools for studying Gray Platelet Syndrome (GPS) pathophysiology through several approaches:

• Phenotypic characterization: Using FITC-conjugated NBEAL2 antibodies to compare protein expression and localization in platelets, megakaryocytes, neutrophils, and mast cells from healthy individuals versus GPS patients or mouse models (Nbeal2^-/-)

• Ultrastructural studies: Combining immunoelectron microscopy with NBEAL2 antibodies to examine subcellular localization and identify specific defects in alpha-granule formation and retention

• Proteomics validation: Confirming mass spectrometry proteomics findings on granule protein deficiencies using immunofluorescence with NBEAL2 and granule-specific protein antibodies

• Hematopoietic differentiation: Monitoring NBEAL2 expression during differentiation of CD34+ hematopoietic stem cells into mature neutrophils or megakaryocytes to identify when NBEAL2-dependent processes become critical

What insights can co-localization studies with NBEAL2 antibodies provide about its molecular interactions?

Co-localization studies using FITC-conjugated NBEAL2 antibodies alongside antibodies for potential interaction partners can reveal:

• Association with RPS6: Recent research identified RPS6 as an interaction partner of NBEAL2, where NBEAL2/RPS6 complex formation appears required for controlling protein homeostasis . Dual-color immunofluorescence can visualize their co-localization in cellular compartments.

• Signaling complexes: NBEAL2 regulates the Shp1-STAT5 signaling axis and the composition of the c-Kit/STAT signalosome in mast cells . Co-staining for these components can map their spatial relationships.

• Vesicular trafficking machinery: Since NBEAL2 contains domains important for vesicle transport, fusion, and protein sorting (BEACH, ConA, WD40, and pleckstrin homology domains) , co-localization with vesicular markers can elucidate its role in granule formation pathways.

• Interactome mapping: Potential interactions with proteins like Dock7, Sec16a, and Vac14, which function in signaling and have been suggested as NBEAL2 interactors , can be visualized through co-staining approaches.

How can NBEAL2 antibodies help distinguish between different cellular phenotypes in hematological disorders?

NBEAL2 antibodies can differentiate cellular phenotypes in hematological disorders through:

• Neutrophil granule analysis: Using FITC-conjugated NBEAL2 antibodies to distinguish between normal neutrophils and those with specific granule deficiencies (as seen in GPS) . Research has shown that neutrophils from GPS patients have normal distribution of azurophilic granules but show deficiency of specific granules, which can be detected using immunofluorescence approaches.

• Platelet classification: Identifying platelets with alpha-granule deficiencies versus those with normal granule content. The intensity of NBEAL2 staining correlates with alpha-granule content, allowing researchers to classify platelets based on their granule status .

• Mast cell differentiation markers: NBEAL2 regulates the expression of transcription factors IRF8, GATA2, and MITF in mast cells . Co-staining for these factors alongside NBEAL2 can help classify mast cell maturation states and abnormalities.

• Multiparameter flow cytometry: Combining FITC-conjugated NBEAL2 antibodies with markers for cell lineage, activation state, and granule content enables high-dimensional analysis of heterogeneous cell populations in blood disorders.

How can inconsistent staining patterns with NBEAL2 antibodies be resolved?

When encountering inconsistent staining patterns with NBEAL2 antibodies, consider these approaches:

• Fixation optimization: Different fixation methods affect epitope accessibility. For NBEAL2, which is associated with membrane structures and granules, compare methanol fixation (which better preserves protein epitopes) with paraformaldehyde fixation (which better preserves cellular structures).

• Antigen retrieval: For IHC-P applications, test different antigen retrieval methods (heat-induced vs. enzymatic) as NBEAL2 epitopes may be masked during tissue processing.

• Permeabilization adjustment: Since NBEAL2 is involved in intracellular granule formation, optimize permeabilization conditions (varying detergent types and concentrations) to ensure antibody access to intracellular compartments.

• Batch variation: When comparing NBEAL2 staining across experiments, use standard positive control samples (like HepG2 cells) to normalize for batch-to-batch antibody variations .

• Genetic variation: In patient samples, inconsistent staining may reflect genetic heterogeneity. GPS can result from different mutations in NBEAL2, potentially affecting antibody binding depending on the epitope recognized .

How should researchers interpret differential NBEAL2 expression across cell types and disease states?

Interpreting differential NBEAL2 expression requires several considerations:

What are the most significant contradictions in published NBEAL2 research findings and how might they be explained?

Several contradictions exist in NBEAL2 research that can affect antibody-based studies:

• Neutrophil phenotypes: Initial cohorts of Nbeal2^-/- mice showed differences in neutrophil counts and mean platelet volumes that were not confirmed in later cohorts. These discrepancies may be due to strain background effects or loosely linked passenger mutations that were removed by consecutive backcrossing . When using NBEAL2 antibodies to characterize neutrophils, researchers should consider genetic background effects.

• Quantification methods: Disagreements in absolute neutrophil counts (ANC) between automated hematology analyzers and flow cytometry have been reported, with evidence that some analyzers overestimate neutrophil counts. Flow cytometry using Trucount beads (CV = 3.48%) is considered more accurate . Researchers should be aware of these methodological differences when correlating NBEAL2 antibody staining with neutrophil counts.

• Function in different cell types: While NBEAL2's role in platelet alpha-granule formation is well-established, its function in mast cells and neutrophils appears more complex and sometimes contradictory. In mast cells, NBEAL2 regulates transcription factor expression and signaling pathways beyond granule formation , while in neutrophils, it affects specific granule content but not azurophilic granules . These differences highlight the need for cell-type specific controls when using NBEAL2 antibodies.

• Human vs. mouse phenotypes: Some features of human GPS are not perfectly recapitulated in mouse models. While taking into account the smaller size of mouse platelets, Nbeal2^-/- platelets show similar morphological characteristics as human GPS including vacuoles, but some subtle differences exist . These species-specific differences should be considered when using NBEAL2 antibodies across model systems.

How can NBEAL2 antibodies be used to investigate NET formation in neutrophils?

Recent research has revealed that neutrophils from GPS patients show defective Neutrophil Extracellular Trap (NET) formation . FITC-conjugated NBEAL2 antibodies can be used to investigate this process through:

• Temporal analysis: Time-course experiments tracking NBEAL2 localization during NET formation using live-cell imaging with FITC-conjugated antibodies or fixed-cell immunofluorescence at defined timepoints.

• Co-localization with NET components: Dual-color immunofluorescence studying NBEAL2 co-localization with histones, granule-derived proteases, and antimicrobial peptides during NET formation.

• Mechanistic studies: Investigating the relationship between NBEAL2, specific granule content, and NET formation by correlating NBEAL2 expression/localization with ROS formation and elastase activity, which are known to be critical for NET formation.

• Comparative analysis: Using NBEAL2 antibodies to compare neutrophils from GPS patients, Nbeal2^-/- mice, and controls to determine if NET formation defects are consistent across species and directly related to NBEAL2 function.

What role does NBEAL2 play in regulating protein homeostasis and how can antibodies help investigate this function?

Recent research has identified that NBEAL2 interacts with RPS6 to control protein homeostasis . FITC-conjugated NBEAL2 antibodies can help investigate this function through:

• Proximity ligation assays: Using NBEAL2 antibodies in conjunction with RPS6 antibodies in proximity ligation assays to visualize and quantify their interactions in different cell types and under various conditions.

• Co-immunoprecipitation validation: Using NBEAL2 antibodies for co-immunoprecipitation experiments to validate interaction partners identified through proteomics approaches.

• Stress response studies: Tracking NBEAL2 localization and expression during cellular stress conditions that affect protein homeostasis (heat shock, oxidative stress, ER stress) to determine if NBEAL2's role extends to stress response mechanisms.

• Ribosomal association: Investigating whether NBEAL2 associates with ribosomes or other translation machinery through co-localization studies with markers of protein synthesis.

What are the best methods for quantifying NBEAL2 expression in immunofluorescence studies?

For accurate quantification of NBEAL2 expression using FITC-conjugated antibodies:

• Standardized acquisition parameters: Maintain consistent exposure times, gain settings, and offset values across all samples to ensure comparable fluorescence intensities.

• Reference standards: Include calibration beads with known fluorescence intensities to normalize fluorescence measurements across experiments.

• Multi-parameter analysis: When examining cell-specific effects, combine NBEAL2 staining with lineage markers to ensure accurate gating and cell identification.

• 3D analysis: For cells with complex morphology (like megakaryocytes), use z-stack acquisitions and 3D reconstruction to capture the total NBEAL2 expression rather than single optical sections.

• Relative quantification approaches: When comparing NBEAL2 expression between conditions, consider using the intensity of platelet staining on a scale (light=1, medium=2, dark=3) as demonstrated in Table 4 of reference :

How can researchers distinguish between primary NBEAL2 defects and secondary effects in cell function studies?

When using NBEAL2 antibodies to study cellular dysfunction:

• Rescue experiments: Express wildtype NBEAL2 in cells from GPS patients or Nbeal2^-/- mice to determine if phenotypes can be reversed, confirming they are directly due to NBEAL2 deficiency rather than secondary adaptations.

• Domain-specific constructs: Express individual domains of NBEAL2 (BEACH, ConA, WD40, pleckstrin homology) to determine which are essential for specific functions, using FITC-conjugated antibodies that recognize specific domains.

• Temporal studies: Use inducible knockout systems to acutely deplete NBEAL2 and distinguish immediate effects (likely primary) from those that develop over time (potentially secondary).

• Cross-system validation: Compare phenotypes between patient cells, mouse models, and CRISPR/Cas9-edited cell lines to identify consistent NBEAL2-dependent effects versus system-specific outcomes.

• Pathway inhibitors: Use specific inhibitors of pathways regulated by NBEAL2 (such as STAT5 inhibitors in mast cells ) to determine if phenotypes can be rescued without restoring NBEAL2 function.

How can NBEAL2 antibodies complement genetic and proteomic analyses in hematological research?

FITC-conjugated NBEAL2 antibodies provide valuable complementary data to genetic and proteomic approaches:

• Genotype-phenotype correlations: For GPS patients with different NBEAL2 mutations, NBEAL2 antibodies can reveal if protein expression, stability, or localization correlates with mutation type and clinical severity.

• Protein interaction validation: Proteomic studies have identified potential NBEAL2 interaction partners like RPS6, Dock7, Sec16a, and Vac14 . FITC-conjugated NBEAL2 antibodies can validate these interactions through co-localization and proximity ligation assays.

• Expression quantification: While RNA sequencing provides transcript-level data, NBEAL2 antibodies enable quantification of protein expression, revealing potential post-transcriptional regulation.

• Structural insights: The predicted structure of NBEAL2 suggests functions in vesicle transport and protein sorting . Immunofluorescence with domain-specific antibodies can test structure-based hypotheses about NBEAL2 function.

• Single-cell analysis: Combining single-cell RNA sequencing with immunofluorescence using NBEAL2 antibodies can correlate transcriptional profiles with protein expression at the individual cell level, revealing heterogeneity within populations.

What are the implications of NBEAL2 research for understanding broader mechanisms of granule biogenesis?

NBEAL2 research using specific antibodies provides insights into fundamental biological processes:

• Cross-lineage comparison: Using FITC-conjugated NBEAL2 antibodies to compare its role in platelets, neutrophils, and mast cells reveals common principles and cell-specific aspects of granule formation.

• Evolutionary conservation: Studying NBEAL2 function across species (human patients vs. mouse models) highlights evolutionarily conserved mechanisms in granule biogenesis.

• Disease mechanisms: Comparing NBEAL2-related granule defects with other granule disorders (Chediak-Higashi syndrome, Hermansky-Pudlak syndrome) can identify common pathways and specific mechanisms.

• Therapeutic targets: Identifying key interactions and pathways regulated by NBEAL2 may reveal potential therapeutic targets for disorders of granule formation and function.

• Fundamental cell biology: NBEAL2's involvement in vesicle transport, membrane fusion, and protein sorting contributes to our understanding of basic cellular processes beyond hematological systems.