NTSR2 Antibody, Biotin conjugated

What is NTSR2 and what are its key biological functions?

NTSR2 is a seven-transmembrane G protein-coupled receptor that has been identified as a driver of apoptosis resistance in leukemic B cells. NTSR2 remains in a constitutively active state in these cells due to its interaction with the oncogenic tyrosine kinase receptor TRKB . This interaction activates survival signaling pathways, notably the SRC and AKT kinase pathways, and increases the expression of anti-apoptotic proteins BCL-2 and BCL-XL . In the central nervous system, NTSR2 is expressed in hippocampal astrocytes, and its expression increases with astrocyte reactivity following neurological insults such as status epilepticus . The NTSR2 gene is located on the short arm of chromosome 2, and gain of this region has been associated with poor prognosis in chronic lymphocytic leukemia (CLL) .

What experimental techniques can detect NTSR2-protein interactions?

Several techniques have proven effective for studying NTSR2 interactions:

• Co-immunoprecipitation assays: These can be used to study the NTSR2-TRKB interaction by pull-down experiments using tagged proteins .

• AlphaScreen technology: This proximity-based assay utilizes streptavidin-donor microbeads that recognize biotin-tagged molecules and anti-IgG-acceptor microbeads bound by specific antibodies . When the donor and acceptor beads are brought into proximity through molecular interactions, a detectable signal is produced.

• Confocal microscopy with dual immunohistochemical labeling: This technique allows visualization of NTSR2 expression patterns and co-localization with other markers such as GFAP for astrocytes .

How is NTSR2 expression quantified in tissue samples?

Quantification of NTSR2 in tissue samples typically involves:

• Immunohistochemistry with fluorescent labeling: NTSR2 protein expression can be visualized using specific antibodies and quantified through mean fluorescence intensity measurements .

• Z-stack confocal microscopy: This approach allows precise determination of co-expression of NTSR2 with cell-type specific markers .

• Image analysis software: Tools like ZEN (Zeiss) and ImageJ (NIH) can be used to quantify immunolabeling by converting images to binary black and white format and applying fixed intensity thresholds .

For accurate quantification of NTSR2 levels in GFAP-positive cells, researchers typically:

• Measure mean fluorescence intensity in specific brain regions

• Identify cells through co-labeling with cell-type specific markers

• Subtract background fluorescence from measurements

• Normalize data to controls

How can researchers optimize pull-down assays for NTSR2-TRKB interaction studies?

To optimize pull-down assays for studying NTSR2-TRKB interactions:

• Design appropriate tagging strategies: The AviTag™ system has been successfully used for biotinylation of NTSR2, allowing for efficient pull-down with streptavidin-coated magnetic beads .

• Co-transfection approach: HEK293T cells can be co-transfected with a plasmid encoding biotin transferase BirA and plasmids encoding the proteins of interest (TRKB and NTSR2-AviTag™) .

• Biotin supplementation: Add biotin to the culture medium post-transfection to enable biotinylation of the AviTag™ by BirA .

• Ligand enhancement: Treatment with BDNF (brain-derived neurotrophic factor), the natural ligand of TRKB, enhances the NTSR2-TRKB interaction, which should be considered in experimental design .

• Quantification: Western blot analysis followed by densitometry can be used to calculate interaction ratios (e.g., TRKB/NTSR2) .

What controls should be included when studying NTSR2-protein interactions?

Essential controls for NTSR2 interaction studies include:

• Empty vector controls: Transfection with empty vectors to control for non-specific binding .

• Domain deletion controls: Using truncated versions of NTSR2 with specific domains removed can help identify interaction interfaces . Research has shown that deletion of the C-terminal end of NTSR2 (NTSR2–ΔID4) significantly reduces co-immunoprecipitation of TRKB, indicating this region's importance in the interaction .

• Antibody specificity controls: For double immunohistochemical labeling with antibodies from the same host species, test specificity by:

  • Incubating sections with antibodies targeting different structures

  • Omitting the second primary antibody while including its corresponding secondary antibody

  • Using normal IgG controls

• Background subtraction: For fluorescence quantification, measure and subtract background from areas devoid of stained cells in the same sections .

How can researchers design peptides targeting NTSR2-protein interactions?

The design of peptides that target NTSR2-protein interactions involves several critical steps:

• Identification of interaction domains: Through systematic domain deletion studies, researchers determined that the C-terminal end of NTSR2 (ID4) is crucial for interaction with TRKB .

• Peptide design: Based on the identified interaction domain, peptides can be designed to mimic the binding interface and competitively inhibit protein-protein interactions .

• Cell penetration enhancement: Addition of cell-penetrating sequences, such as TAT peptide, significantly improves cellular uptake. Research showed that TAT-Pep-V5 peptide effectively penetrated CLL-B cells, while Pep-V5 alone showed limited penetration .

• Validation of target binding: Verify that the designed peptide binds to the intended target protein (e.g., TRKB) and disrupts the target interaction (e.g., NTSR2-TRKB) .

• Functional assessment: Evaluate the peptide's effect on downstream signaling pathways and cellular phenotypes to confirm its biological activity .

What are the challenges in detecting NTSR2 in different cell types?

Detecting NTSR2 across different cell types presents several challenges:

• Cell-type specific expression patterns: NTSR2 shows differential expression across tissues. In the brain, NTSR2 immunolabeling patterns are region- and laminar-specific within the hippocampus, with variations in the stratum oriens, stratum radiatum, stratum lacunosum-moleculare, and the dentate gyrus .

• Subcellular localization: NTSR2 shows both membrane and cytoplasmic localization. In astrocytes, NTSR2 is expressed in cell bodies and processes, with patterns changing from diffuse in control conditions to more punctiform following pathological stimuli .

• Expression changes during pathological states: NTSR2 expression increases with astrocyte reactivity following insults such as status epilepticus, requiring different detection sensitivities for baseline versus upregulated states .

• Background and non-specific binding: When performing immunohistochemistry, background fluorescence must be carefully measured in areas devoid of stained cells and subtracted from intensity measurements .

How does biotin conjugation affect antibody performance in NTSR2 detection?

Biotin conjugation offers several advantages for NTSR2 detection but requires specific considerations:

• Streptavidin-biotin interaction: The strong affinity between biotin and streptavidin (or avidin) enables sensitive detection and pull-down of biotinylated NTSR2 antibodies or tagged NTSR2 proteins .

• Amplification systems: The biotin-streptavidin system allows for signal amplification in detection methods such as AlphaScreen technology, where streptavidin-donor microbeads interact with biotinylated molecules .

• Enzymatic biotinylation: The BirA biotin ligase system enables site-specific biotinylation of AviTag™-fused proteins, providing precise control over the biotinylation process for pull-down experiments .

• Potential interference: Endogenous biotin can potentially interfere with detection systems, particularly in tissues with high biotin content, requiring appropriate blocking steps.

What methods can validate NTSR2 antibody specificity?

Validation of NTSR2 antibody specificity is crucial and can be accomplished through:

• Western blot analysis: Confirm antibody specificity by detecting a band of the expected molecular weight for NTSR2 .

• Knockdown/knockout controls: Compare antibody staining between wild-type samples and those with reduced or eliminated NTSR2 expression.

• Peptide competition assays: Pre-incubation of the antibody with the immunizing peptide should reduce or eliminate specific staining .

• Multiple antibody comparison: Use different antibodies targeting distinct epitopes of NTSR2 to confirm staining patterns .

• Cross-reactivity testing: Test the antibody against related receptors (e.g., NTSR1, NTSR3) to ensure specificity for NTSR2.

How can NTSR2 antibodies be utilized in cancer research?

NTSR2 antibodies have significant applications in cancer research, particularly in studying chronic lymphocytic leukemia (CLL):

• Oncogenic pathway analysis: NTSR2 antibodies can be used to study the NTSR2-TRKB oncogenic pathway, which activates survival signaling in leukemic B cells .

• Therapeutic target validation: Studies have demonstrated that the NTSR2-TRKB interaction represents a promising therapeutic target in CLL, and antibodies can help validate this potential .

• Biomarker development: Given that NTSR2 is highly expressed in leukemic B cells but not in B cells from healthy donors, antibodies can potentially be used to develop diagnostic or prognostic biomarkers .

• Drug development support: NTSR2 antibodies can help screen and validate therapeutic peptides or small molecules designed to disrupt the NTSR2-TRKB interaction .

What is the role of NTSR2 in neurological conditions and how can it be studied?

NTSR2 plays significant roles in neurological conditions that can be investigated using specific techniques:

• Astrocyte reactivity marker: NTSR2 expression increases in hippocampal astrocytes following status epilepticus, indicating its potential role in neuroinflammation .

• Cell-type specific analysis: Dual immunohistochemical labeling with NTSR2 and GFAP antibodies allows identification of NTSR2 expression in astrocytes versus other cell types .

• Subcellular localization changes: Following pathological stimuli, NTSR2 distribution changes from diffuse to punctiform in astrocytes, which can be visualized using high-resolution confocal microscopy .

• Quantitative analysis: Mean fluorescence intensity measurements of NTSR2 in specific brain regions (dentate gyrus, CA1, CA3) can quantify expression changes during disease progression .

How can researchers optimize NTSR2 detection in fixed tissue samples?

For optimal detection of NTSR2 in fixed tissue samples:

• Fixation protocols: Proper fixation is crucial for preserving NTSR2 epitopes. Paraformaldehyde fixation has been successfully used for NTSR2 immunodetection in brain tissue .

• Antigen retrieval: Depending on the fixation method, antigen retrieval steps may be necessary to expose NTSR2 epitopes.

• Double immunolabeling: For co-localization studies with markers that use antibodies from the same host species (e.g., NTSR2 and GFAP rabbit antibodies), sequential immunolabeling protocols with appropriate blocking steps are essential .

• Confocal microscopy settings: Use of Z-stack function helps determine precise co-expression of NTSR2 with other markers in the same cells .

• Image analysis: Convert images to 16-bit black and white format and apply fixed intensity thresholds for consistent quantification across samples .

What emerging technologies might enhance NTSR2 research?

Several emerging technologies hold promise for advancing NTSR2 research:

• CRISPR-Cas9 gene editing: Precise modification of NTSR2 or associated genes could provide new insights into its functions and interactions.

• Single-cell approaches: Single-cell RNA sequencing and proteomics can reveal cell-type specific expression patterns and heterogeneity in NTSR2 signaling.

• Advanced imaging techniques: Super-resolution microscopy could provide more detailed visualization of NTSR2 subcellular localization and interactions.

• Targeted protein degradation: Techniques like PROTACs (Proteolysis Targeting Chimeras) could offer new ways to modulate NTSR2 levels with greater specificity than conventional inhibitors.

• Machine learning applications: AI-based approaches could help identify novel NTSR2 interaction partners and signaling pathways from large datasets.

How might NTSR2 antibodies contribute to therapeutic development?

NTSR2 antibodies can contribute to therapeutic development in several ways:

• Target validation: Antibodies can confirm the role of NTSR2 in disease processes, validating it as a therapeutic target .

• Mechanism studies: Antibodies help elucidate the mechanisms by which NTSR2 contributes to diseases, informing rational drug design .

• Companion diagnostics: NTSR2 antibodies could potentially be developed as companion diagnostics to identify patients likely to respond to NTSR2-targeted therapies.

• Therapeutic antibody development: While the current research focuses on peptide inhibitors of NTSR2-TRKB interaction , antibodies targeting NTSR2 could potentially be developed as therapeutic agents themselves.

• Response monitoring: Antibodies can help monitor changes in NTSR2 expression or localization in response to therapy.