NTSR1 Antibody, HRP conjugated

What is NTSR1 and what are its molecular characteristics?

NTSR1 (Neurotensin Receptor 1) is a G-protein coupled receptor that specifically binds the tridecapeptide neurotensin (NTS). It functions through G proteins that activate a phosphatidylinositol-calcium second messenger system, initiating downstream signaling cascades. When activated, NTSR1 triggers pathways that protect cells against apoptosis through the activation of MAP kinases . The receptor exists in multiple molecular forms, primarily due to post-translational modifications rather than alternative splicing variants.

Research has identified three distinct protein forms of NTSR1: NTSR1-high (a glycosylated form), NTSR1-low (an N-terminally cleaved form), and NTSR1-LP (a low prevalence form) . These various forms appear simultaneously in cell systems expressing NTSR1 and demonstrate different molecular weights when analyzed via SDS-PAGE. The complexity of these multiple forms presents significant challenges for antibody-based detection methods, requiring careful experimental design and appropriate controls.

Understanding the molecular biology of NTSR1 is critical for experimental design, particularly regarding antibody selection and validation strategies. NTSR1 is particularly significant in cancer research, with high expression being a negative prognostic marker in stage I lung adenocarcinomas and ductal invasive carcinomas .

What advantages do HRP-conjugated NTSR1 antibodies offer over unconjugated alternatives?

HRP (Horseradish Peroxidase) conjugation provides several methodological advantages for NTSR1 detection in research applications. While unconjugated antibodies require a secondary detection system, HRP-conjugated antibodies enable direct detection through enzymatic conversion of substrate, streamlining experimental workflows and potentially reducing non-specific background signals that can arise from secondary antibody interactions.

The enzymatic amplification properties of HRP significantly enhance detection sensitivity, which is particularly valuable when studying NTSR1 in tissues or cell types with naturally low expression levels. The signal amplification occurs through HRP's catalytic conversion of substrates to produce either colorimetric, chemiluminescent, or fluorescent signals depending on the detection system employed. This amplification capability allows researchers to detect NTSR1 present at physiologically relevant concentrations that might be below the detection threshold of direct fluorescent conjugates.

How do researchers distinguish between different NTSR1 molecular forms using antibodies?

Distinguishing between the three NTSR1 forms (NTSR1-high, NTSR1-low, and NTSR1-LP) represents a significant technical challenge. Most commercially available anti-NTSR1 antibodies yield multiple bands in Western blot analysis, making it difficult to differentiate between specific NTSR1 forms and non-specific signals . Researchers have developed several approaches to overcome this limitation.

One effective strategy involves isolating total cell membranes and extracting the GPCR fraction prior to SDS-PAGE analysis, which substantially reduces non-specific signals . Validation through genetic approaches is essential - using siRNA, shRNA, or CRISPR-Cas9 knockdown controls allows researchers to conclusively identify which bands correspond to genuine NTSR1 signals. For instance, generating NTSR1-depleted clones (e.g., HT29-KD) provides an excellent negative control for antibody specificity testing .

An alternative method involves creating tagged NTSR1 variants (e.g., GFP-tagged or FLAG-tagged NTSR1) and detecting these with corresponding anti-tag antibodies. Studies have confirmed that anti-GFP and anti-FLAG antibodies targeting tagged NTSR1 detect the same three molecular weight species identified by anti-NTSR1 antibodies . It's worth noting that while NTSR1-FLAG resolves at the same apparent molecular weight as non-tagged NTSR1, NTSR1-GFP demonstrates a mobility shift of approximately 27 kDa corresponding to the GFP molecular weight .

What are the optimal Western blot protocols for detecting multiple NTSR1 forms?

Optimizing Western blot protocols for NTSR1 detection requires addressing several critical parameters to differentiate between the three molecular forms while minimizing non-specific signals. Based on published methodologies, a comprehensive approach begins with careful sample preparation. Isolating total cell membranes and extracting the GPCR fraction substantially improves signal specificity compared to using whole cell lysates . This fractionation step is particularly important when working with endogenous NTSR1 rather than overexpressed systems.

For protein separation, standard SDS-PAGE using 8-10% polyacrylamide gels effectively resolves the different NTSR1 forms (NTSR1-high at approximately 50-60 kDa, NTSR1-low at 37-45 kDa, and NTSR1-LP at lower molecular weights) . Complete protein denaturation is essential for consistent results - samples should be heated at 95°C for 5 minutes in Laemmli buffer containing SDS and reducing agents. Including positive controls (cells with verified NTSR1 expression) and negative controls (NTSR1-depleted cells generated through CRISPR-Cas9 or shRNA knockdown) in each experiment is critical for band identification .

For electroblotting, using PVDF membranes rather than nitrocellulose often provides better retention of membrane proteins like NTSR1. The blocking step requires careful optimization - while 5% non-fat milk is commonly used, it may contain glycosylated proteins that potentially cross-react with certain antibodies. BSA-based blocking solutions (3-5%) represent a suitable alternative for NTSR1 detection. When using HRP-conjugated primary antibodies, researchers should extend incubation times (overnight at 4°C) and optimize antibody concentrations to balance specific signal detection against background.

How should immunohistochemistry protocols be optimized for NTSR1 detection in tissue samples?

Immunohistochemical detection of NTSR1 in tissue samples requires careful protocol optimization due to the receptor's variable expression levels and multiple molecular forms. Based on validated protocols, formalin-fixed paraffin-embedded (FFPE) tissue sections represent the standard sample preparation method, with antigen retrieval being a critical step for successful NTSR1 detection . Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is generally effective, though the optimal conditions may vary depending on the specific anti-NTSR1 antibody used.

For NTSR1 antibody application, dilution ratios between 1:100 and 1:200 have shown effective results in rat brain tissue sections . When using HRP-conjugated primary antibodies, researchers should increase the stringency of washing steps to minimize background staining. The detection system significantly impacts sensitivity - DAB (3,3'-diaminobenzidine) substrate provides a stable, permanent signal suitable for archival samples, while tyramide signal amplification can enhance detection sensitivity for low-abundance NTSR1 expression .

Methodological validation through appropriate controls is essential: positive controls should include tissues with confirmed NTSR1 expression (e.g., specific brain regions), while negative controls should involve either omission of primary antibody or preferably the use of tissues from NTSR1 knockout models. For multiplex immunohistochemistry applications, research indicates that NTSR1 antibodies may be successfully combined with markers for cellular compartments or signaling partners, provided that primary antibodies are raised in different host species to prevent cross-reactivity .

What considerations are important when selecting NTSR1 antibodies for specific epitopes?

Epitope selection represents a critical decision point when choosing NTSR1 antibodies for specific research applications. Current commercial antibodies target various regions of the NTSR1 protein, with those recognizing the N-terminus, C-terminus, or extracellular loops having distinct advantages depending on the experimental context . Antibodies targeting the N-terminal region (AA 1-67) are particularly useful for detecting full-length NTSR1, but may fail to recognize the NTSR1-low form which lacks this region due to N-terminal cleavage .

For applications requiring detection of all NTSR1 forms, antibodies targeting the second extracellular loop (AA 209-224) or internal regions (AA 188-290) have demonstrated reliable results across multiple detection methods including Western blotting, immunohistochemistry, and immunofluorescence . These regions appear to remain intact across the various NTSR1 forms, providing consistent epitope availability even after post-translational modifications.

When examining NTSR1 localization at the cell surface, antibodies targeting extracellular domains are preferable since they can detect the receptor without cell permeabilization in flow cytometry or immunofluorescence applications . Research has demonstrated successful plasma membrane NTSR1 detection using antibodies against extracellular epitopes in non-permeabilized HEK293T-NTSR1-GFP cells .

For HRP-conjugated antibodies specifically, epitope accessibility becomes even more critical due to the steric hindrance potentially introduced by the enzyme conjugate. Internal region epitopes may demonstrate reduced detection efficiency with direct HRP conjugates compared to unconjugated primary antibodies followed by HRP-labeled secondary antibodies.

Why do researchers observe multiple bands when performing Western blot analysis with NTSR1 antibodies?

The presence of multiple bands in NTSR1 Western blots represents one of the most common technical challenges encountered by researchers. This pattern results from a combination of biological and methodological factors. Biologically, NTSR1 exists naturally as three distinct molecular forms: NTSR1-high (glycosylated), NTSR1-low (N-terminally cleaved), and NTSR1-LP (low prevalence form) . These forms appear simultaneously in expressing cells and demonstrate different electrophoretic mobilities during SDS-PAGE.

Non-specific binding of commercial antibodies further complicates band interpretation. Most commercially available anti-NTSR1 antibodies yield multiple bands, requiring careful validation to distinguish between genuine NTSR1 signals and non-specific interactions . Non-specific bands around 50 kDa are particularly common with many commercial NTSR1 antibodies . Signal validation through genetic knockdown approaches (siRNA, shRNA, or CRISPR-Cas9) represents the gold standard for band identification.

Post-translational modifications also contribute to band pattern complexity. NTSR1-high carries N-glycosylation modifications that increase its apparent molecular weight . Treatment with glycosylation inhibitors like tunicamycin reduces NTSR1-high expression and generates interim glycosylation states appearing as multiple lower bands . O-glycosylation inhibitors like BADG similarly affect banding patterns by disrupting normal glycosylation processing .

To minimize these issues, researchers should implement specialized sample preparation techniques. Isolating total cell membranes and extracting the GPCR fraction before SDS-PAGE substantially reduces non-specific signals compared to using whole cell lysates . Including both positive controls (verified NTSR1-expressing cells) and negative controls (NTSR1-knockdown cells) in each experiment facilitates accurate band identification.

How can researchers address cross-reactivity issues with NTSR1 antibodies?

Cross-reactivity represents a significant concern when working with NTSR1 antibodies, particularly when studying tissues or cells expressing multiple neurotensin receptor subtypes. The neurotensin receptor family includes NTSR1, NTSR2, and NTSR3/sortilin, with NTSR1 and NTSR3 both binding neurotensin with high affinity . Traditional 125I-NTS binding assays cannot distinguish between NTSR1 and NTSR3 expression, necessitating antibody-based approaches for specific detection .

Several methodological strategies can minimize cross-reactivity issues. Epitope selection represents the first critical decision point - antibodies targeting unique regions of NTSR1 with minimal sequence homology to other neurotensin receptors demonstrate higher specificity. Antibodies recognizing the second extracellular loop (AA 209-224) or internal regions (AA 188-290) have shown reliable selectivity for NTSR1 .

Validation through genetic approaches provides definitive confirmation of antibody specificity. Researchers should generate negative controls using CRISPR-Cas9, siRNA, or shRNA techniques to knockdown NTSR1 expression . These controls allow unambiguous identification of non-specific signals. Alternative validation strategies include creating tagged NTSR1 variants (GFP-tagged or FLAG-tagged) and detecting these with anti-tag antibodies, which eliminates concerns about cross-reactivity with other neurotensin receptor subtypes .

For immunohistochemical applications, cross-adsorption of antibodies against recombinant proteins representing potential cross-reactive targets (NTSR2, NTSR3) can improve specificity. When using HRP-conjugated antibodies specifically, optimizing antibody concentration is particularly important, as higher concentrations may increase non-specific interactions. Titration experiments should determine the minimum antibody concentration that provides adequate specific signal while minimizing background.

What approaches help differentiate between glycosylated and non-glycosylated forms of NTSR1?

Distinguishing between glycosylated (NTSR1-high) and non-glycosylated forms (NTSR1-low) of NTSR1 requires specific experimental approaches that target the post-translational modifications. Research has established that NTSR1-high carries N-glycosylation modifications that increase its apparent molecular weight, while NTSR1-low does not carry glycosylation modifications . Several methodological strategies can effectively differentiate these forms.

Enzymatic deglycosylation treatments provide direct evidence of glycosylation status. Treatment with PNGase F, which specifically cleaves N-linked glycans, converts NTSR1-high to a lower molecular weight form without affecting NTSR1-low . This differential response confirms the glycosylation status of each band. Similarly, treatment with Endo H, which removes only high mannose and some hybrid types of N-linked glycans, helps determine the glycan complexity on NTSR1-high .

Pharmacological inhibition of glycosylation pathways offers another approach. Treatment with tunicamycin, which blocks the first step of N-glycosylation, decreases NTSR1-high expression while causing the appearance of multiple lower molecular weight bands representing interim glycosylation states . BADG (benzyl-2-acetoamino-2-deoxy-α-D-galactopyranoside), an O-glycosylation inhibitor, produces similar effects by disrupting the cellular glycosylation machinery .

Lectin blotting provides complementary evidence by directly detecting glycans on Western blots. Different lectins (e.g., ConA for high-mannose N-glycans, WGA for terminal N-acetylglucosamine) can identify specific glycan structures on NTSR1-high. For proteomic confirmation, mass spectrometry analysis following immunoprecipitation with anti-NTSR1 antibodies can precisely characterize the glycosylation sites and structures present on NTSR1-high versus NTSR1-low.

How can NTSR1 antibodies contribute to understanding receptor trafficking and localization?

NTSR1 antibodies serve as essential tools for investigating receptor trafficking and subcellular localization dynamics. Research has demonstrated that NTSR1 undergoes biosynthesis through the secretory pathway, including glycosylation initiation in the endoplasmic reticulum, maturation in the Golgi apparatus, and trafficking to the plasma membrane . Antibody-based approaches have been instrumental in visualizing these processes.

Immunofluorescence microscopy using NTSR1 antibodies has revealed colocalization of NTSR1 with the Golgi marker GM130 in PANC1 cells overexpressing NTSR1-GFP, confirming trafficking through this compartment . Further studies demonstrated colocalization with peripheral F-actin, strongly suggesting plasma membrane localization . For definitive confirmation of cell surface expression, flow cytometry with anti-NTSR1 antibodies on non-permeabilized cells provides quantitative data on membrane-localized receptor populations .

Agonist-induced receptor dynamics can be monitored through antibody-based approaches. Treatment with neurotensin (NTS) induces a decrease in NTSR1-high form with concomitant increase in NTSR1-low form in HEK293T-NTSR1 cells, suggesting agonist-promoted conversion between forms . In contrast, in PANC1 and HT29#3 cells, NTS treatment decreases NTSR1-low form, indicating receptor degradation pathways . These differential responses highlight cell-type specific regulation mechanisms that can be captured through careful antibody-based analysis.

For advanced applications, researchers have developed pulse-chase immunoprecipitation protocols using anti-NTSR1 antibodies to track newly synthesized receptor populations through the secretory pathway. Combining antibody-based detection with subcellular fractionation techniques allows quantitative assessment of receptor distribution across cellular compartments under various stimulus conditions. These approaches provide mechanistic insights into how NTSR1 signaling is regulated through controlled receptor trafficking.

What role does NTSR1 play in cancer research and how can antibodies advance this field?

NTSR1 has emerged as a significant factor in cancer progression, particularly in breast and lung cancers. Research has established that NTSR1 high expression serves as a negative prognostic marker in stage I lung adenocarcinomas treated by surgery alone and in ductal invasive carcinomas . Both neurotensin (NTS) and NTSR1 are expressed in approximately 20% of breast tumors and 40% of lung tumors, suggesting widespread relevance in cancer biology .

Functional studies using genetic approaches have demonstrated that removal of NTSR1 expression in both lung and breast cancer cells reduces tumor growth and metastasis, establishing the direct contribution of this receptor to tumor progression . Mechanistically, NTS/NTSR1 signaling drives cancer aggressiveness through multiple pathways. Sustained stimulation of NTSR1 results in the activation of matrix metalloproteinase 1 (MMP1), release of HB-EGF and NRG1, and subsequent overexpression and activation of EGFR, HER2, and HER3 . This cascade ultimately enhances experimental lung tumor growth.

Antibody-based methodologies have been crucial for advancing this research area. Immunohistochemical studies using anti-NTSR1 antibodies have established expression patterns across tumor tissues and correlated these with patient outcomes . Western blot analysis has revealed complex interactions between NTSR1 and growth factor receptor systems, showing that NTS/NTSR1 signaling increases HER2 and HER3 protein levels (and to a lesser extent, EGFR levels) without affecting their mRNA expression . This suggests that NTSR1 signaling alters receptor recycling and degradation pathways rather than transcriptional regulation.

For future cancer research, NTSR1 antibodies will enable high-throughput screening of patient-derived samples to identify individuals who might benefit from NTSR1-targeted therapies. Developing therapeutic antibodies that block NTS/NTSR1 interactions could potentially disrupt the downstream signaling cascades that promote tumor growth and metastasis.

How can post-translational modifications of NTSR1 be systematically studied using specific antibodies?

Post-translational modifications (PTMs) significantly influence NTSR1 function, and antibody-based approaches offer powerful tools for their systematic investigation. Research has established that NTSR1 undergoes various modifications, most notably N-glycosylation to form NTSR1-high and proteolytic N-terminal cleavage to generate NTSR1-low . Experimental strategies using specific antibodies can elucidate these complex modifications.

Modification-specific antibodies represent an emerging approach for direct PTM detection. While standard NTSR1 antibodies recognize all forms of the receptor, developing antibodies that specifically recognize either glycosylated epitopes or neo-epitopes created by proteolytic cleavage would enable direct visualization of modified receptor populations. These tools would facilitate quantitative assessment of how various stimuli or cellular conditions affect the ratio of modified to unmodified receptor.

Immunoprecipitation combined with mass spectrometry provides comprehensive PTM mapping. Anti-NTSR1 antibodies can isolate the receptor from cellular lysates, followed by proteomic analysis to identify specific modification sites and structures. This approach has revealed that NTSR1 undergoes N-glycosylation and N-terminal cleavage, but potentially harbors additional modifications that remain uncharacterized . Researchers can apply similar strategies to investigate phosphorylation, ubiquitination, or other potential modifications that might regulate receptor function.

Site-directed mutagenesis coupled with antibody detection offers mechanistic insights. By creating NTSR1 variants with mutations at potential modification sites and analyzing their expression patterns using anti-NTSR1 antibodies, researchers can determine which residues are critical for specific modifications. This approach helped establish that NTSR1 undergoes N-glycosylation rather than alternative processing .

For temporal analysis of modification dynamics, pulse-chase experiments combined with sequential immunoprecipitation using different NTSR1 antibodies can track the conversion between modified forms following stimulation or during receptor maturation. These approaches collectively provide a comprehensive toolkit for dissecting the complex PTM landscape governing NTSR1 biology.