NOTCH2 Antibody, FITC conjugated

What is NOTCH2 and why is it important in research?

NOTCH2 is a transmembrane receptor encoded by the NOTCH2 gene in humans. It is a 2471-amino acid protein that functions as a member of the NOTCH family of receptors. Notch2 is a documented neural stem cell marker with predicted cellular localization in the cytoplasm, nucleus, and membrane compartments. The protein contains multiple glycosylation sites that affect its function .

In research, NOTCH2 is crucial because it participates in highly conserved signaling pathways that regulate cell fate decisions during development and homeostasis. Dysregulation of NOTCH2 signaling has been implicated in various pathological conditions, including cancer, developmental disorders, and immune system abnormalities. For instance, antibodies targeting the negative regulatory region (NRR) of NOTCH2 have been used to reverse osteopenic phenotypes in mouse models of Hajdu Cheney syndrome, which features a Notch2 gain-of-function mutation .

How does FITC conjugation affect NOTCH2 antibody performance in flow cytometry?

• Signal intensity: FITC has a relatively lower quantum yield compared to newer fluorophores, which may affect sensitivity when detecting low-expression targets.

• Spectral characteristics: FITC has excitation/emission peaks around 495/519 nm, which may overlap with other common fluorophores or cellular autofluorescence.

• Photobleaching: FITC is more susceptible to photobleaching than newer fluorophores, requiring careful handling and minimized light exposure.

• pH sensitivity: FITC fluorescence is optimal at neutral to basic pH conditions; acidic environments can significantly reduce signal strength.

To optimize performance when detecting NOTCH2 expression, researchers should consider cell-specific expression levels. For example, studies have shown that Notch2 surface expression varies significantly across B cell subsets, with approximately 2-fold upregulation in reporter+CD38+CD95+ non-GCB cells compared to reporter− B cells, while CD38−CD95+ GCB cells progressively downregulate Notch2 surface expression over time .

What are the validated applications for NOTCH2 antibody in research?

Based on the available information, NOTCH2 antibodies have been validated for numerous applications in research:

• Flow cytometry: Used to quantify and characterize Notch2 expression on various cell populations, particularly in studying B cell differentiation and tracking expression changes during immune responses .

• Functional assays (FA): Anti-Notch neutralizing antibodies have been used in functional assays to block Notch signaling .

• Neutralization studies (Neut): Antibodies targeting the negative regulatory region (NRR) of Notch2 have been employed to lock the receptor in its quiescent state, preventing activation .

• Immunohistochemistry: For visualizing Notch2 expression in tissue sections, allowing researchers to track the location of Notch2-expressing cells, such as B cells migrating toward the marginal zone .

• Cellular localization studies: Since Notch2 can be found in cytoplasmic, nuclear, and membrane-associated locations, specific antibodies have been developed to detect these different pools .

For optimal results, researchers should select antibodies that have been validated specifically for their application of interest and experimental system.

How can researchers distinguish between cis and trans NOTCH2 signaling interactions?

Distinguishing between cis (same-cell) and trans (adjacent cell) NOTCH2 signaling interactions represents a significant challenge in Notch biology research. Based on systematic studies of Notch signaling interactions, the following methodological approaches are recommended:

• Engineered cell line systems: Develop stable cell lines that provide quantitative readouts of Notch signaling activity, receptor level, and ligand level. CHO-K1 cells are ideal as they exhibit negligible endogenous expression of Notch receptors and ligands .

• Reporter constructs: Use chimeric human Notch2 receptors with the intracellular domain replaced by a minimal transcription factor (e.g., Gal4) and a fluorescent protein reporter (e.g., H2B-mTurq2) to measure receptor expression. Implement a UAS promoter driving expression of another fluorescent protein (e.g., H2B-mCitrine) to reflect Notch activity .

• Controlled ligand expression: Engineer sender cells with precise control over ligand expression levels, using fluorescent protein markers to quantify expression .

• Normalization approaches: Quantify signaling by normalizing reporter fluorescence by receptor expression, and further normalizing by ligand expression to control for variation across sender populations .

• Pharmacological inhibition: Use γ-secretase inhibitors like DAPT to differentiate between different modes of activation. This approach can help distinguish genuine cis-activation from artifacts of intercellular signaling .

Recent research has revealed striking differences in cis-interactions among receptor-ligand combinations. For example, Delta ligands cis-activate Notch2 much more strongly than Notch1, all four ligands cis-inhibit Notch1, and Jagged (but not Delta) ligands cis-inhibit Notch2 .

What considerations should be made when using NOTCH2 antibodies in B cell differentiation studies?

When studying B cell differentiation using NOTCH2 antibodies, researchers should consider:

• Dynamic expression patterns: Notch2 surface expression varies significantly throughout B cell differentiation. In vitro studies show approximately 20-fold upregulation of Notch2 on follicular B (FoB) cells upon combined CD40 and BCR stimulation, peaking between 24-48 hours. In vivo, Notch2 is upregulated in non-germinal center B (non-GCB) cells but progressively downregulated in germinal center B (GCB) cells .

• Transcription factor interactions: Notch2 signaling influences the expression of key transcription factors. While control/CAR+ B cells strongly induce Bcl6 expression but maintain low Irf4 levels (consistent with GCB phenotype), N2IC/hCD2+ B cells do not upregulate Bcl6 but show strong induction of Irf4 .

• Differentiation pathway tracking: Notch2 signaling guides B cells away from germinal centers. Some antigen-activated Notch2-expressing B cells differentiate into marginal zone B (MZB) cells. Approximately one-fourth of N2IC/hCD2+Irf4+B220+ cells display MZB cell phenotype by day 7 post-immunization, with frequencies increasing over time .

• Surface marker co-expression: Track multiple surface markers simultaneously (CD21, CD23, CD1d, IgM, CD38) to accurately identify MZB cell phenotypes (CD23lowCD21highIgMhighCD1dhighCD38high) that emerge through Notch2 signaling .

• Localization studies: Combine flow cytometry with spatial analysis, as Notch2-expressing cells migrate toward the marginal zone over time, eventually forming a marginal zone ring around the follicle by day 30 .

• Genetic validation: Use Notch2 knockout models (N2KO) to confirm the Notch2-dependency of observed phenotypes. For example, reporter+CD23lowCD21high B cells are significantly reduced in immunized N2KO//CAR mice compared to controls .

What approaches help differentiate between active and inactive NOTCH2 signaling?

Distinguishing between active and inactive NOTCH2 signaling requires sophisticated experimental approaches:

• Transcriptional reporters: Implement reporter systems where fluorescent protein expression is driven by Notch-responsive elements. Systems using the Gal4 transcription factor and UAS promoter driving H2B-mCitrine expression have successfully quantified Notch activity .

• Cleavage-specific antibodies: Use antibodies that specifically recognize the cleaved intracellular domain of Notch2 (N2IC) to directly detect activated Notch2.

• Antibodies targeting the negative regulatory region (NRR): These antibodies can lock Notch2 in its quiescent state by binding to an epitope that bridges the LIN-12 Notch repeat and heterodimerization domain. This approach has been used therapeutically and can serve as a tool to prevent Notch activation .

• Downstream target analysis: Measure the expression of established Notch2 target genes (e.g., Hes1, Hey1) using RT-qPCR or single-cell RNA sequencing.

• Pharmacological inhibition: Use γ-secretase inhibitors like DAPT to block S3 cleavage and compare signaling in treated versus untreated conditions. Since DAPT prevents S3 but not S2 receptor cleavage, this approach can help distinguish between different modes of activation .

• Combined surface and intracellular staining: Use flow cytometry to simultaneously detect surface Notch2 expression and intracellular transcription factors influenced by Notch signaling, such as Irf4 and Bcl6 in B cells .

• Mathematical modeling: Implement deterministic mathematical modeling approaches to quantify the dynamics of Notch2 signaling during cellular differentiation processes .

What controls should be included when using FITC-conjugated NOTCH2 antibodies in flow cytometry?

When using FITC-conjugated NOTCH2 antibodies in flow cytometry, researchers should include the following controls:

• Isotype control: Include a FITC-conjugated antibody of the same isotype as the NOTCH2 antibody but with no specificity for mammalian antigens to assess non-specific binding.

• Fluorescence minus one (FMO) control: Include all fluorophores in your panel except FITC to establish proper gating boundaries and account for spectral overlap.

• Unstained control: Include completely unstained cells to establish baseline autofluorescence.

• Positive control: Include a cell type known to express high levels of NOTCH2, such as activated follicular B cells which show approximately 20-fold upregulation of Notch2 surface expression upon CD40- and BCR-stimulation .

• Negative control: Use cells with known low or absent NOTCH2 expression, such as CHO-K1 cells which exhibit negligible endogenous expression of Notch receptors .

• Blocking control: Pre-incubate a sample with unconjugated NOTCH2 antibody before adding the FITC-conjugated version to confirm specificity.

• Compensation controls: Single-stained controls for each fluorophore in your panel to properly compensate for spectral overlap.

• Genetic knockout or knockdown control: When possible, include NOTCH2-deficient cells (N2KO) to confirm antibody specificity .

• Expression validation control: Consider parallel staining with a different clone of NOTCH2 antibody conjugated to a different fluorophore to confirm expression patterns.

• Fringe enzyme expression control: If studying Notch signaling modifications, include controls for Fringe enzyme expression, which can be achieved through siRNA knockdown followed by plasmid transfection of wild-type or catalytically inactive mutants .

How can researchers optimize staining protocols for detecting both membrane and nuclear NOTCH2?

Detecting both membrane and nuclear NOTCH2 requires a carefully optimized protocol that preserves epitopes while allowing antibody access to different cellular compartments:

• Fixation optimization:

  • For membrane NOTCH2: Use a mild fixative like 2% paraformaldehyde for 10-15 minutes at room temperature to preserve surface epitopes.

  • For total NOTCH2 (membrane and nuclear): Use a sequential fixation approach with 2% paraformaldehyde followed by methanol or a commercial fixation/permeabilization kit optimized for nuclear antigens.

• Permeabilization strategy:

  • For membrane-only staining: Avoid detergents or use very mild permeabilization (0.1% saponin).

  • For nuclear staining: Use stronger permeabilization with 0.1-0.3% Triton X-100 or specialized nuclear permeabilization buffers.

• Epitope retrieval: For fixed tissues or cells with significant cross-linking, consider antigen retrieval methods such as heat-induced epitope retrieval in citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0).

• Sequential staining approach:

  • First stain for membrane NOTCH2 using surface staining protocols

  • Then fix and permeabilize cells more stringently

  • Finally stain for intracellular/nuclear NOTCH2

• Antibody selection: Choose antibodies that recognize either the extracellular domain (for membrane) or the intracellular domain (for nuclear localization) of NOTCH2. The antibody directed to the negative regulatory region (NRR) of Notch2 would be suitable for membrane detection .

• Blocking optimization: Use comprehensive blocking (5-10% serum plus Fc receptor blocking) to reduce background, particularly important for intracellular staining.

• Detection strategy: Consider using different fluorophores for membrane versus nuclear NOTCH2 to simultaneously visualize both pools.

• Validation with functional markers: Co-stain with markers of Notch activation such as target genes or with antibodies specifically recognizing the cleaved intracellular domain.

What considerations should be made when studying NOTCH2 in different B cell populations?

When studying NOTCH2 in different B cell populations, researchers should consider:

How should researchers normalize and quantify NOTCH2 expression in flow cytometry experiments?

Proper normalization and quantification of NOTCH2 expression in flow cytometry experiments is critical for generating reliable and comparable data:

How can researchers distinguish between genuine NOTCH2 signaling and experimental artifacts?

Distinguishing genuine NOTCH2 signaling from experimental artifacts requires careful experimental design and multiple controls:

• Receptor-ligand specificity controls: Since the Notch system includes multiple receptors and ligands with varying specificities, systematically test all receptor-ligand combinations. Studies have shown that signaling strength varies widely across different Notch-ligand-Fringe combinations .

• Pharmacological validation: Use γ-secretase inhibitors like DAPT to distinguish different modes of activation. Since DAPT prevents S3 but not S2 receptor cleavage, researchers can identify potential artifacts from intercellular signaling occurring prior to assay initiation .

• Genetic validation: Use Notch2 knockout models (N2KO) to confirm the Notch2-dependency of observed phenotypes. For example, reporter+CD23lowCD21high B cells are significantly reduced in immunized N2KO//CAR mice compared to controls .

• Cleavage-specific detection: Implement approaches that specifically detect cleaved, active Notch2 intracellular domain rather than total protein.

• Cis- versus trans-activation differentiation: Design experiments to distinguish between cis-activation (same cell) and trans-activation (adjacent cell) signals. For example, research has shown that cis-activation in Delta-Notch2 receivers can rival the strength of trans-activation and is blocked by γ-secretase inhibitors .

• Background signaling assessment: Use appropriate base cell lines, such as CHO-K1 cells, which exhibit negligible endogenous expression of Notch receptors and ligands and no endogenous Notch signaling activity .

• Signaling mode combination analysis: Be aware that cis- and trans-signaling can combine differently across ligand-receptor combinations. Some combinations show increased activity with both cis- and trans-interactions, while others show preference for a particular signaling mode .

• Reproducibility across models: Validate findings in multiple experimental systems, including cell lines, primary cells, and in vivo models.

What approaches help interpret contradictory results in NOTCH2 signaling studies?

Interpreting contradictory results in NOTCH2 signaling studies requires careful consideration of multiple factors:

How can researchers address sensitivity limitations of FITC-conjugated NOTCH2 antibodies?

FITC-conjugated antibodies may present sensitivity challenges due to the fluorophore's properties. Researchers can address these limitations through several approaches:

• Signal amplification strategies:

  • Implement biotin-streptavidin systems for enhanced signal

  • Use tyramide signal amplification (TSA) for dramatically increased sensitivity

  • Consider multi-layer detection with primary antibody, biotinylated secondary, and streptavidin-FITC

• Instrumentation optimization:

  • Use cytometers with optimal laser/filter combinations for FITC (488nm excitation, 530/30nm bandpass filter)

  • Implement photomultiplier tube (PMT) voltage optimization

  • Consider spectral cytometers with unmixing capabilities for better separation from autofluorescence

• Staining protocol refinements:

  • Increase antibody concentration (after careful titration)

  • Extend incubation times at lower temperatures (4°C overnight)

  • Implement two-step staining approaches with unconjugated primary and FITC-conjugated secondary antibodies

• Sample preparation improvements:

  • Minimize autofluorescence through careful buffer selection

  • Implement quenching of endogenous fluorescence

  • Consider cell surface acid washing to remove non-specific binding

• Alternative approaches for low-expression scenarios:

  • Select brighter fluorophores (PE, APC) for detecting low-level Notch2 expression

  • Use indirect staining methods with biotinylated primary antibodies and streptavidin-conjugated fluorophores

• Target enrichment:

  • Implement immunomagnetic pre-enrichment for Notch2-expressing cells

  • Use stimulation protocols that upregulate Notch2 expression before analysis, as demonstrated with FoB cells which show ~20-fold upregulation upon combined CD40- and BCR-stimulation

• Data analysis strategies:

  • Use probability binning or other sensitive statistical approaches to detect subtle shifts

  • Implement fluorescence minus one (FMO) controls for accurate gating

  • Consider using the stain index rather than mean fluorescence intensity for comparisons

What are the best practices for storing and handling FITC-conjugated NOTCH2 antibodies?

To maintain optimal performance of FITC-conjugated NOTCH2 antibodies, researchers should follow these best practices:

• Storage conditions:

  • Store at 4°C (short-term) or -20°C (long-term) in the dark

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

  • Consider adding protein stabilizers (1% BSA) and preservatives (0.09% sodium azide) for long-term storage

  • Monitor and maintain recommended pH conditions (typically pH 7.2-7.4)

• Light protection:

  • Store in amber vials or wrap containers in aluminum foil

  • Minimize exposure to light during all handling steps

  • Work under reduced ambient lighting

  • Use amber microcentrifuge tubes for dilutions

• Handling precautions:

  • Centrifuge vials briefly before opening to collect liquid at the bottom

  • Mix gently by inversion or finger-tapping rather than vortexing

  • Use low-protein-binding tubes for dilutions

  • Implement aseptic technique to prevent contamination

• Quality control:

  • Periodically test antibody performance using positive control samples

  • Document lot numbers and performance characteristics

  • Consider reference standards for consistent performance evaluation

  • Implement stability indicators (e.g., testing aliquots at defined intervals)

• Reconstitution and dilution:

  • Use only recommended buffers for reconstitution of lyophilized antibodies

  • Prepare fresh working dilutions on the day of experiment

  • Filter-sterilize buffers to remove particles that can cause high background

• Transport considerations:

  • Transport on ice and protected from light

  • Avoid shipping delays that could expose antibodies to temperature fluctuations

  • Consider temperature-logging devices for valuable shipments

• Documentation:

  • Maintain detailed records of receipt date, lot number, aliquoting, and usage

  • Record performance characteristics over time to monitor stability

  • Document freeze-thaw cycles and storage conditions

By implementing these storage and handling practices, researchers can maximize the lifespan and performance of their FITC-conjugated NOTCH2 antibodies, ensuring consistent results across experiments.

How can NOTCH2 antibodies be utilized in studying developmental disorders?

NOTCH2 antibodies offer valuable tools for studying developmental disorders, particularly those involving Notch2 signaling dysregulation:

• Hajdu-Cheney syndrome research: This rare genetic disorder features Notch2 gain-of-function mutations. Antibodies directed to the negative regulatory region (NRR) of Notch2 have successfully reversed the osteopenic phenotype in mouse models by locking the receptor in its quiescent state .

• Developmental pathway analysis: Notch2 antibodies can help map critical developmental decisions during embryogenesis and tissue formation by tracking expression patterns across different cell populations.

• Therapeutic development: Structure-function studies using different Notch2 antibodies can guide the development of targeted therapeutics for disorders involving Notch2 dysregulation.

• Mechanistic investigations: Antibodies that distinguish between active and inactive forms of Notch2 can help elucidate the molecular mechanisms underlying developmental abnormalities.

• In vivo imaging: Labeled Notch2 antibodies can facilitate non-invasive tracking of Notch2-expressing cells during development in appropriate animal models.

• Genetic interaction studies: Combining Notch2 antibody-based detection with genetic manipulations can reveal interactions between Notch2 signaling and other developmental pathways.

What are the emerging applications of NOTCH2 antibodies in cancer research?

Emerging applications of NOTCH2 antibodies in cancer research include:

• Cancer stem cell identification: Since Notch2 is a documented neural stem cell marker , antibodies can help identify and isolate cancer stem cells in various malignancies.

• Signaling pathway inhibition: Neutralizing antibodies targeting the negative regulatory region (NRR) of Notch2 can be used to study the effects of Notch2 inhibition on tumor growth and progression .

• Diagnostic and prognostic applications: Expression patterns of Notch2 across tumor samples can be correlated with clinical outcomes to identify prognostic biomarkers.

• Combination therapy studies: Notch2 antibodies can be used to study the effects of combining Notch pathway inhibition with other therapeutic approaches.

• Tumor microenvironment analysis: Multi-parameter flow cytometry incorporating Notch2 antibodies can characterize the complex interactions between tumor cells and immune components.

• Targeted drug delivery: Notch2 antibodies can potentially be used to develop antibody-drug conjugates for targeted therapy of Notch2-expressing tumors.

• Resistance mechanism studies: Analyzing changes in Notch2 expression and signaling can help understand mechanisms of resistance to conventional therapies.

How might advanced multiplex imaging with NOTCH2 antibodies enhance our understanding of tissue architecture?

Advanced multiplex imaging with NOTCH2 antibodies can significantly enhance our understanding of tissue architecture through:

• Spatial mapping of signaling networks: Multiplexed imaging allows visualization of Notch2 in relation to its ligands and downstream effectors within intact tissues, revealing spatial organization of signaling networks.

• Cell fate tracking: Combining Notch2 antibodies with lineage markers can reveal how Notch2 signaling influences cell fate decisions in situ, as demonstrated in B cell differentiation studies where Notch2-expressing cells migrate toward the marginal zone over time, eventually forming a marginal zone ring around the follicle .

• Temporal-spatial dynamics: Time-course studies with in vivo imaging can capture the dynamic nature of Notch2 expression and signaling during development, disease progression, or treatment response.

• Microenvironmental context: Multiplexed imaging can reveal how Notch2 signaling is influenced by the local tissue microenvironment, including interactions with extracellular matrix components and neighboring cells.

• Single-cell resolution in tissue context: Technologies like imaging mass cytometry or co-detection by indexing (CODEX) allow simultaneous detection of dozens of markers, including Notch2, at single-cell resolution while maintaining tissue architecture information.

• 3D reconstruction: Advanced imaging modalities can generate 3D reconstructions of Notch2 expression patterns throughout tissue volumes, providing insights into complex architectural features.

• Computational analysis integration: Machine learning algorithms can identify spatial patterns and cellular neighborhoods associated with specific Notch2 expression profiles, revealing emergent properties not observable through conventional analyses.