NTSR2 Antibody, HRP conjugated

What is NTSR2 and why is it an important research target?

NTSR2 (Neurotensin Receptor type 2) is a G protein-coupled receptor that has emerged as a critical biological target across multiple research domains. It plays essential roles in both normal physiology and pathological conditions. In B-cell chronic lymphocytic leukemia (B-CLL), NTSR2 functions as a driver of apoptosis resistance, being highly expressed in B-CLL cells while its natural ligand neurotensin (NTS) shows minimal expression . Additionally, NTSR2 has been implicated in neurological processes and has been studied in glioblastoma cell lines where it's associated with the internalization-dependent activation of ERK 1/2 . The receptor's versatile biological roles make it a valuable research target for both cancer biology and neuroscience investigations.

What is the molecular characterization of human NTSR2?

Human NTSR2 (UniProtID: O95665) is a G protein-coupled receptor characterized by its seven transmembrane domains. The protein includes specific regions like the amino acid sequence 359-410 that are commonly used as immunogens for antibody production . The NTSR2 gene can undergo alternative splicing, resulting in variant forms such as vNTSR2, which has been documented in rat models . At the functional level, NTSR2 can exist in a constitutively active phosphorylated state, particularly in pathological conditions like B-CLL, where this activation is not dependent on its natural ligand neurotensin but rather results from interactions with other receptor proteins like TrkB .

How does NTSR2 differ from NTSR1 in biological systems?

NTSR2 and NTSR1 exhibit distinct expression patterns and functional roles across different biological systems. In B-CLL cells, NTSR2 is significantly overexpressed (30-fold higher than in normal B lymphocytes), while NTSR1 remains undetectable at both mRNA and protein levels . This differential expression suggests specialized roles for each receptor subtype. Functionally, NTSR2 has been linked to apoptosis resistance in cancer cells through activation of survival pathways involving Src, JNK, p38, and Akt, leading to increased expression of anti-apoptotic proteins like Bcl-xL and Bcl-2 . The two receptors also differ in their ligand binding properties, with NTSR2 sometimes referred to as the "levocabastine-sensitive neurotensin receptor" . These distinctions are crucial when designing experiments targeting specific neurotensin receptor subtypes.

What are the key specifications of NTSR2 Antibody, HRP conjugated?

The NTSR2 Antibody, HRP conjugated (e.g., SKU: A30112) is a polyclonal antibody raised in rabbits using recombinant Human Neurotensin receptor type 2 protein (amino acids 359-410) as the immunogen . This antibody demonstrates reactivity specifically to human NTSR2 and is primarily validated for ELISA applications. The technical specifications include:

This detailed characterization ensures researchers can properly incorporate this reagent into their experimental protocols .

What advantages does HRP conjugation provide for NTSR2 antibody applications?

HRP (Horseradish Peroxidase) conjugation offers several methodological advantages for NTSR2 antibody applications in research settings. The direct conjugation eliminates the need for secondary antibody incubation steps, thereby reducing protocol time, background signal, and cross-reactivity issues. This streamlined approach is particularly beneficial in ELISA, where the HRP conjugate can directly catalyze chromogenic or chemiluminescent substrate reactions for quantitative detection .

The HRP enzyme maintains high catalytic activity even after conjugation to antibodies, providing excellent sensitivity for detecting low-abundance NTSR2 expression. This is especially valuable when investigating normal physiological levels of NTSR2, which may be considerably lower than the overexpression observed in pathological states like B-CLL . Additionally, the established nature of HRP-based detection systems means they are compatible with widely available substrates and detection instruments, making this conjugated antibody adaptable to various laboratory settings.

How should researchers optimize storage conditions for NTSR2 Antibody, HRP conjugated?

Proper storage of NTSR2 Antibody, HRP conjugated is critical for maintaining reagent integrity and experimental reproducibility. Upon receipt, the antibody should be stored at either -20°C or -80°C according to manufacturer recommendations . The inclusion of 50% glycerol in the buffer formulation helps prevent freeze-thaw damage, but researchers should nonetheless minimize freeze-thaw cycles as repeated temperature fluctuations can compromise both antibody binding capacity and HRP enzymatic activity.

For working solutions, aliquoting the stock antibody into single-use volumes is strongly recommended. When preparing these aliquots, researchers should use sterile microcentrifuge tubes and aseptic technique to prevent microbial contamination. Short-term storage (1-2 weeks) of working dilutions at 4°C is possible due to the preservative (0.03% Proclin 300) in the buffer, but extended storage should always utilize freezing conditions. Before each use, researchers should allow the antibody to equilibrate to room temperature completely and mix gently by inversion rather than vortexing, which can damage antibody structure through shear forces.

What protocols are recommended for using NTSR2 Antibody, HRP conjugated in ELISA applications?

For ELISA applications using NTSR2 Antibody, HRP conjugated, researchers should implement a systematic optimization approach. A recommended protocol begins with coating microplate wells with the target antigen (recombinant NTSR2 or cell lysates containing NTSR2) at concentrations ranging from 0.1-10 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C. After blocking with 2-5% BSA or non-fat milk in PBS-T (PBS with 0.05% Tween-20) for 1-2 hours at room temperature, apply the HRP-conjugated NTSR2 antibody at dilutions ranging from 1:500 to 1:5000 in blocking buffer for 1-2 hours at room temperature .

Following thorough washing with PBS-T (at least 4-5 washes of 3-5 minutes each), add appropriate HRP substrate (TMB, ABTS, or chemiluminescent reagents) and develop according to substrate specifications. For quantitative analysis, include a standard curve using purified recombinant NTSR2 protein. The optimal antibody dilution should be determined empirically for each experimental system, with 1:1000 serving as a reasonable starting point for most applications. When comparing NTSR2 expression between different samples (e.g., normal B cells versus B-CLL cells), ensure equal protein loading and include appropriate positive and negative controls .

Can NTSR2 Antibody, HRP conjugated be adapted for immunohistochemistry despite not being explicitly validated for this application?

While NTSR2 Antibody, HRP conjugated is primarily validated for ELISA, researchers interested in immunohistochemistry (IHC) applications may adapt the reagent through careful methodological modifications and validation. When attempting this cross-application, consider that unconjugated NTSR2 antibodies targeting similar epitopes have shown successful application in IHC . For adaptation, researchers should first optimize antigen retrieval methods (heat-induced epitope retrieval using citrate buffer pH 6.0 or EDTA buffer pH 9.0) to ensure proper exposure of the target epitope.

The HRP conjugation provides a direct detection advantage, eliminating the need for secondary antibodies, but may require signal amplification through tyramide signal amplification (TSA) systems for optimal sensitivity. Initial validation should include positive controls (tissues known to express NTSR2, such as specific brain regions or B-CLL samples) and negative controls (NTSR2-knockout tissues or isotype controls) . Concentration gradients should be tested, typically starting at higher concentrations (1:50-1:200) than those used for ELISA. Critical validation steps must include pre-absorption controls with recombinant NTSR2 protein and comparison with alternative validated NTSR2 antibodies to confirm staining specificity.

How can researchers distinguish between NTSR2 and its splice variant vNTSR2 when using this antibody?

Distinguishing between NTSR2 and its splice variant vNTSR2 requires careful experimental design since the HRP-conjugated antibody may recognize epitopes common to both forms. Based on research in rat models where both variants have been identified in glioma cells and astroglia , researchers should first determine whether the antibody's target epitope (amino acids 359-410 in human NTSR2) is preserved or altered in the splice variant. This can be accomplished through sequence analysis and epitope mapping.

For experimental differentiation, implementing a multi-method approach is recommended. RT-PCR should be used first to confirm the presence of both transcripts, which can be distinguished by their different sizes (600 bp for NTSR2 and 418 bp for vNTSR2 in rat models) . For protein detection, researchers should complement antibody-based detection with methods that can separate proteins by size, such as Western blotting, where full-length NTSR2 and truncated vNTSR2 would show different migration patterns. If both variants are present, quantitative analyses using the HRP-conjugated antibody alone may not accurately represent the distribution of specific variants, necessitating the use of variant-specific antibodies or probes as complementary approaches.

How should researchers interpret NTSR2 expression levels when comparing normal and pathological tissues?

When comparing NTSR2 expression between normal and pathological tissues, researchers should implement a comprehensive analytical framework that accounts for biological and technical variables. Studies have shown that NTSR2 is significantly overexpressed in B-CLL cells (30-fold higher) compared to normal B lymphocytes , establishing a precedent for substantial expression differences in pathological states. Quantitative analysis should employ multiple technical replicates (minimum n=3) and biological replicates representing population diversity.

For accurate interpretation, researchers should:

• Normalize NTSR2 expression to validated housekeeping genes or proteins that remain stable across compared conditions

• Consider the expression of NTSR2's binding partners, particularly TrkB, which has been shown to interact with NTSR2 and influence its activity

• Assess both mRNA (through qRT-PCR) and protein levels, as post-transcriptional regulation may create discrepancies

• Evaluate the phosphorylation state of NTSR2, as its constitutive activation has been observed in certain pathological conditions independent of ligand binding

Statistical analysis should employ appropriate tests based on data distribution, with multiple comparison corrections when analyzing various tissue types simultaneously. Results should be interpreted within the biological context, considering that NTSR2 expression patterns may vary independently of common disease markers (as observed in B-CLL, where NTSR2 expression doesn't correlate with markers like TP53 deletion or IGHV mutation) .

What are common sources of background signal when using NTSR2 Antibody, HRP conjugated, and how can they be minimized?

Background signal issues with HRP-conjugated antibodies like the NTSR2 Antibody can arise from multiple sources and require systematic troubleshooting. Endogenous peroxidase activity in tissue samples or cells can generate false-positive signals, particularly in samples rich in peroxidase-containing cells like leukocytes. This can be mitigated by including a peroxidase quenching step (3% hydrogen peroxide in methanol for 10-15 minutes) before primary antibody application .

Non-specific binding represents another major source of background. The polyclonal nature of the antibody increases the risk of binding to proteins sharing similar epitopes. Researchers should optimize blocking procedures using 3-5% BSA or normal serum from the same species as the secondary antibody (if using a detection system requiring secondaries). Increasing the concentration of blocking agent and extending blocking time (2-3 hours at room temperature or overnight at 4°C) may improve specificity.

Additional strategies include:

• Increasing washing duration and frequency (5-6 washes of 5 minutes each)

• Optimizing antibody dilution through titration experiments

• Adding 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

• Including 0.1-0.5M NaCl in antibody diluent to reduce ionic interactions

• Pre-absorbing the antibody with unrelated proteins to remove promiscuous binding

For particularly challenging samples, signal-to-noise ratio can be improved by using specialized detection systems like polymer-HRP technologies or implementing biotin-streptavidin amplification systems followed by carefully controlled development times .

How can researchers validate the specificity of NTSR2 Antibody, HRP conjugated in their experimental system?

Validating the specificity of NTSR2 Antibody, HRP conjugated requires a multi-faceted approach to ensure experimental rigor. The gold standard for specificity validation involves parallel testing in NTSR2 knockout models, such as the NTS2 null mice described in the literature , compared with wild-type counterparts. When knockout models are unavailable, researchers should implement RNA interference approaches, using siRNA pools directed against NTSR2 mRNA similar to those described in B-CLL studies .

Additional validation strategies include:

• Peptide competition assays: Pre-incubating the antibody with excess recombinant NTSR2 protein (specifically the 359-410AA immunogen region) should abolish or significantly reduce specific signals

• Cross-validation with alternative antibodies: Comparing detection patterns using antibodies targeting different NTSR2 epitopes can confirm specificity

• Correlation with mRNA expression: Demonstrating concordance between protein detection using the antibody and mRNA levels measured by qRT-PCR

• Heterologous expression systems: Testing the antibody in cell lines engineered to overexpress NTSR2 versus control transfected cells

• Western blot analysis: Confirming that the antibody detects a protein of the expected molecular weight (~45 kDa for NTSR2)

Documentation of these validation steps enhances experimental reproducibility and strengthens the reliability of research findings involving NTSR2 detection.

How can NTSR2 Antibody, HRP conjugated be utilized in studying NTSR2-TrkB interactions in cancer cells?

The study of NTSR2-TrkB protein interactions represents an advanced application area where NTSR2 Antibody, HRP conjugated can provide valuable insights. Research has demonstrated that NTSR2 and TrkB physically interact in B-CLL cells, with this interaction being enhanced upon BDNF (Brain-Derived Neurotrophic Factor) stimulation . To investigate this interaction, researchers can implement co-immunoprecipitation (co-IP) protocols where cell lysates are precipitated with anti-TrkB antibodies, followed by detection of co-precipitated NTSR2 using the HRP-conjugated NTSR2 antibody in Western blot analysis.

For in situ visualization of these interactions, proximity ligation assays (PLA) can be adapted using the HRP-conjugated NTSR2 antibody in combination with appropriate anti-TrkB antibodies. This would allow quantification of interaction events at the single-cell level. Researchers could further examine the functional consequences of these interactions by combining antibody-based detection with phosphorylation-specific antibodies to monitor activation of downstream signaling pathways like ERK1/2, p38, JNK, and Akt .

Experimental designs should include appropriate controls such as BDNF stimulation, which has been shown to promote NTSR2-TrkB interaction, and TrkB inhibitors to confirm specificity . Through such approaches, researchers can correlate NTSR2-TrkB interaction with cellular phenotypes like apoptosis resistance in various cancer models beyond B-CLL.

What methodological approaches can be used to study the constitutive activation of NTSR2 independent of its natural ligand?

Investigating the constitutive activation of NTSR2 independent of neurotensin binding requires sophisticated methodological approaches focusing on receptor phosphorylation and conformational states. Research has revealed that NTSR2 can exist in a constitutively active phosphorylated state in B-CLL cells, likely resulting from interaction with TrkB rather than ligand binding . To study this phenomenon, researchers should implement phosphorylation-specific detection methods, potentially developing phospho-specific NTSR2 antibodies that can be used in conjunction with the total NTSR2 antibody, HRP conjugated.

Flow cytometry offers a powerful approach to quantify the proportion of phosphorylated versus total NTSR2 across different cell populations, while phosphoproteomics analysis can identify specific phosphorylation sites mediating activation. NTSR2 activation can also be assessed functionally by measuring:

• G-protein coupling using [35S]GTPγS binding assays or BRET-based G-protein activation sensors

• β-arrestin recruitment through BRET or enzyme complementation assays

• Internalization kinetics using antibody feeding assays

• Downstream signaling pathway activation (ERK1/2, p38, JNK, Akt)

Intervention studies employing NTSR2-specific inhibitors or siRNA-mediated knockdown provide critical evidence of receptor-specific effects, while mutagenesis of potential phosphorylation sites can identify residues essential for constitutive activation. Together, these approaches can elucidate the unconventional activation mechanisms of NTSR2 in pathological contexts.

How can NTSR2 Antibody, HRP conjugated be integrated into high-throughput screening of potential NTSR2 inhibitors for cancer therapy?

The development of NTSR2 inhibitors represents a promising therapeutic strategy, particularly for B-CLL where NTSR2 overexpression contributes to apoptosis resistance . NTSR2 Antibody, HRP conjugated can be integrated into high-throughput screening (HTS) workflows through the development of cell-based ELISA systems for rapid evaluation of compounds that modulate NTSR2 expression, phosphorylation, or protein-protein interactions.

A methodological framework for such screening might include:

• Primary screen: Cell-based ELISA measuring NTSR2 expression/phosphorylation levels in response to compound libraries

  • Plate B-CLL cells or NTSR2-overexpressing model cells in 384-well plates

  • Treat with compound libraries at standardized concentrations

  • Fix and permeabilize cells

  • Detect total NTSR2 using HRP-conjugated antibody

  • In parallel plates, detect phosphorylated NTSR2 using phospho-specific primary antibodies

• Secondary screening:

  • Measure anti-apoptotic protein expression (Bcl-2, Bcl-xL) in hits from primary screen

  • Assess cell viability and apoptosis markers (Annexin V/PI staining, DNA fragmentation)

  • Evaluate NTSR2-TrkB interaction through co-IP or PLA methods

• Tertiary screening:

  • Determine effects on downstream signaling pathways (ERK1/2, p38, JNK, Akt)

  • Test efficacy in patient-derived B-CLL samples

  • Assess selectivity across NTSR subtypes

This systematic approach leverages the specific detection capabilities of NTSR2 Antibody, HRP conjugated to facilitate the discovery of compounds that could potentially reverse the apoptosis resistance conferred by NTSR2 in malignancies like B-CLL.

How does the performance of NTSR2 Antibody, HRP conjugated compare with other detection methods for NTSR2 research?

Comparative analysis with alternative methods reveals:

The optimal method depends on research questions and available resources. For high-throughput screening or routine quantification, HRP-conjugated antibodies excel, while spatial distribution studies benefit from fluorescent approaches. Research examining NTSR2 at the functional level should complement antibody-based detection with functional assays like calcium mobilization or G-protein activation measures .

What are the current knowledge gaps regarding NTSR2 function that could be addressed using NTSR2 Antibody, HRP conjugated?

Despite significant advances in NTSR2 research, several critical knowledge gaps remain that could be addressed using well-validated NTSR2 antibodies. The HRP-conjugated format is particularly suitable for quantitative analyses across diverse biological contexts. Major unresolved questions include:

• Tissue-specific expression patterns: While NTSR2 overexpression has been documented in B-CLL and certain neural tissues, comprehensive profiling across human tissues and disease states remains incomplete. Systematic immunohistochemical studies could map NTSR2 distribution across normal tissues and pathological samples.

• Regulatory mechanisms controlling NTSR2 expression: The factors governing NTSR2 upregulation in malignancies like B-CLL remain poorly understood. ChIP-based approaches combined with expression analysis could identify transcription factors and epigenetic modifications regulating NTSR2 expression.

• NTSR2 heterodimerization partners beyond TrkB: While interaction with TrkB has been documented , NTSR2 may form functional complexes with additional partners. Proteomic analysis of NTSR2 immunoprecipitates could identify novel interaction networks.

• Post-translational modifications: Beyond phosphorylation, other modifications like glycosylation, ubiquitination, or SUMOylation may regulate NTSR2 function and trafficking. Specialized immunoprecipitation protocols followed by mass spectrometry could characterize these modifications.

• NTSR2 in immune function: The role of NTSR2 in normal immune cells versus malignant B-CLL cells requires further exploration, particularly regarding potential immunomodulatory functions.

Addressing these gaps would significantly advance understanding of NTSR2 biology and its potential as a therapeutic target.

What emerging technologies could enhance the utility of NTSR2 Antibody, HRP conjugated in neurodegenerative disease research?

Emerging technologies offer opportunities to extend the applications of NTSR2 Antibody, HRP conjugated beyond traditional methodologies, particularly in neurodegenerative disease research where NTSR2 may play unexplored roles. Single-cell technologies represent a frontier for enhanced resolution of NTSR2 expression patterns. Adapting the HRP-conjugated antibody for cyclic immunofluorescence (CycIF) or co-detection by indexing (CODEX) would allow simultaneous detection of NTSR2 alongside dozens of other markers at single-cell resolution within brain tissue sections.

Spatial transcriptomics approaches could be complemented with antibody-based protein detection to correlate NTSR2 protein expression with transcriptional landscapes across brain regions affected in neurodegenerative conditions. For functional studies, optogenetic approaches combined with NTSR2 detection could elucidate activity-dependent regulation of the receptor.

The development of blood-brain barrier (BBB)-penetrant NTSR2-targeted probes derived from the antibody's binding domain could facilitate in vivo imaging of NTSR2 expression using PET or SPECT modalities. This would be particularly valuable given evidence from mouse models suggesting NTSR2's involvement in responses to ethanol , which may have implications for addiction-related neurodegeneration.