OPRL1 Antibody, Biotin conjugated

What is OPRL1 and what is its functional significance in neural tissues?

OPRL1 (opioid related nociceptin receptor 1) is a G-protein coupled receptor that functions as the receptor for the endogenous neuropeptide nociceptin/orphanin FQ, a 17 amino acid peptide . This receptor belongs to the class A (Rhodopsin-like) γ subfamily of G protein-coupled receptors (GPCRs) and shares significant structural homology with μ-, δ- and κ-opioid receptors .

OPRL1 is widely expressed throughout the central nervous system, with particularly high levels in the cortex, hippocampus, amygdala, hypothalamus, central grey, pontine nuclei, interpeduncular nucleus, substantia nigra, raphe complex, locus coeruleus, and spinal cord . The receptor plays essential roles in:

• Nociception and pain perception

• Cognitive functions

• Modulation of inflammation and immune responses

• Regulation of locomotor activity

• Anxiety and depression

• Food intake and obesity

• Learning and memory processes

• Addiction mechanisms

• Cardiovascular homeostasis

• Intestinal motility

At the cellular level, OPRL1 activation triggers intracellular signaling that includes inhibition of adenylyl cyclase, activation of phospholipase C, modulation of ion channels (K⁺ channel activation and Ca²⁺ channel inhibition), and activation of mitogen-activated protein kinases (MAPKs) . The primary functional consequence in neural tissues is the inhibition of neurotransmitter release, including acetylcholine, catecholamines, GABA, glutamate, 5-hydroxytryptamine, and tachykinins .

What are the key applications for biotin-conjugated OPRL1 antibodies in research?

Biotin-conjugated OPRL1 antibodies offer versatility across multiple applications due to the strong and specific binding between biotin and streptavidin/avidin systems. The primary applications include:

Biotin conjugation is particularly advantageous for multiplex detection systems, where multiple targets can be visualized simultaneously. The biotin-streptavidin interaction offers high affinity (Kd ≈ 10⁻¹⁵ M), providing robust binding that enhances detection sensitivity compared to unconjugated antibodies .

What is the molecular profile of OPRL1 and how does it affect antibody selection?

OPRL1 has specific molecular characteristics that researchers should consider when selecting appropriate antibodies:

When selecting OPRL1 antibodies, researchers should consider the specific epitope recognized. For example, the antibody described in the Alomone Labs data (AOR-015) targets amino acid residues 337-352 of rat OPRL1 at the intracellular C-terminus , while other antibodies may target different regions. This affects the antibody's performance in different applications, particularly when protein conformation or post-translational modifications are important experimental considerations.

What are the recommended storage and handling conditions for maintaining OPRL1 antibody activity?

Proper storage and handling of OPRL1 antibodies are critical for maintaining their activity and specificity:

For biotin-conjugated antibodies specifically, it's important to store them protected from light to prevent photobleaching of the biotin moiety. Additionally, researchers should be aware that sodium azide can inhibit horseradish peroxidase (HRP) activity, which may impact certain detection methods. When using HRP-based detection systems, ensure the final working concentration of sodium azide is below 0.1% .

How should optimal conditions be established for immunohistochemical detection of OPRL1?

Optimizing immunohistochemical detection of OPRL1 requires careful consideration of several experimental parameters:

For biotin-conjugated OPRL1 antibodies specifically, researchers should be aware of endogenous biotin in certain tissues, particularly liver, kidney, and brain. Pre-blocking with avidin-biotin blocking kits is essential to prevent false-positive signals. Additionally, positive controls using tissues with known OPRL1 expression (such as human gliomas tissue or mouse brain tissue) should be included , alongside negative controls where the primary antibody is omitted or pre-absorbed with the immunizing peptide.

The experimental conditions have been validated for detecting OPRL1 in human gliomas tissue and mouse brain tissue, with optimal dilutions ranging from 1:20 to 1:200 for IHC applications . Researchers should perform their own titration experiments to determine the optimal concentration for their specific experimental system.

What are the critical validation strategies for confirming OPRL1 antibody specificity?

Rigorous validation of OPRL1 antibody specificity is essential for reliable research outcomes. Multiple complementary approaches should be employed:

Western blot validation has confirmed detection of OPRL1 in HeLa cells, SH-SY5Y cells, rat brain, mouse brain, and U-87 MG cell lysates . An example validation approach is demonstrated in the Alomone Labs data, where the anti-OPRL1 antibody (AOR-015) shows specific staining at approximately 45 kDa in rat brain, mouse brain, and U-87 MG lysates, with this signal being abolished when the antibody is pre-incubated with the immunizing peptide .

For biotin-conjugated antibodies specifically, additional controls should include testing for endogenous biotin interference by applying only the detection reagent (streptavidin-HRP/AP) without primary antibody to assess background signal levels.

How should experimental design address potential cross-reactivity with other opioid receptors?

OPRL1 shares significant structural homology with other opioid receptors (μ, δ, and κ), creating potential cross-reactivity challenges. Strategic experimental design can address these concerns:

The data indicates that anti-OPRL1 antibodies have been verified in multiple systems, including Western blot analysis of HeLa cells, SH-SY5Y cells, rat brain, mouse brain, and U-87 MG lysates . When designing competitive binding assays or displacement studies, researchers should include controls with ligands specific to other opioid receptors to ensure OPRL1-specific detection.

For immunohistochemical applications, researchers should compare OPRL1 staining patterns with known distribution maps of other opioid receptors. Areas with exclusive OPRL1 expression (such as specific nuclei in the hypothalamus or amygdala) can serve as internal controls for antibody specificity assessment .

What are the optimal approaches for multiplexed detection of OPRL1 with other signaling proteins?

Multiplexed detection of OPRL1 alongside other signaling proteins offers comprehensive insights into receptor interactions and signaling networks. Biotin-conjugated OPRL1 antibodies are particularly valuable in these applications:

When designing multiplexed experiments with biotin-conjugated OPRL1 antibodies, consider these protein interaction partners that have been experimentally linked to OPRL1 signaling:

• G proteins (particularly Gi/Go subtypes)

• Adenylyl cyclase (showing inhibition upon OPRL1 activation)

• Phospholipase C (activated through OPRL1 signaling)

• K⁺ and Ca²⁺ channels (modulated by OPRL1 activation)

• Mitogen-activated protein kinases (activated downstream of OPRL1)

• NF-κB signaling components (involved in OPRL1-mediated autophagy regulation)

For brain tissue studies, multiplexed detection of OPRL1 with neurotransmitter markers (cholinergic, GABAergic, glutamatergic, serotonergic) can provide valuable insights into the receptor's role in specific neural circuits .

How can researchers address common technical challenges with OPRL1 antibody applications?

Researchers frequently encounter technical challenges when working with OPRL1 antibodies. Here are evidence-based approaches to common issues:

For biotin-conjugated antibodies specifically, researchers should be particularly alert to endogenous biotin interference. This is especially relevant in tissues with high metabolic activity like liver, kidney, and certain brain regions. Pre-blocking with an avidin-biotin blocking kit is essential, as is the inclusion of a negative control where only the streptavidin detection reagent is applied .

The data demonstrates that optimal dilution ranges vary significantly by application: 1:500-1:2000 for Western blot, 1:20-1:200 for IHC, and 0.5-4.0 μg for immunoprecipitation . Researchers should perform their own titration experiments when establishing protocols for new experimental systems.

How should researchers interpret OPRL1 expression patterns across different neural tissues?

Interpreting OPRL1 expression patterns requires understanding of the receptor's normal distribution and the factors that influence its expression:

When interpreting immunohistochemical results, researchers should consider cellular localization patterns. As a G-protein coupled receptor, OPRL1 should primarily show membrane localization, though some intracellular staining may represent internalized receptors or newly synthesized protein in the secretory pathway .

Discrepancies between mRNA and protein expression levels may reflect post-transcriptional regulation. Additionally, OPRL1 expression can be dynamically regulated in response to physiological stimuli or pathological conditions . Comparative analysis across different experimental models and validation with multiple detection methods (IHC, Western blot, qPCR) provides the most comprehensive understanding of OPRL1 expression patterns.

What considerations are essential when analyzing co-localization of OPRL1 with other neural markers?

Co-localization studies with OPRL1 require careful technical and analytical considerations to yield reliable results:

Published data indicates that OPRL1 is expressed in multiple neuronal populations and potentially in glial cells . When designing co-localization studies, researchers should consider these potential OPRL1-expressing cell types and appropriate markers:

• Neurons: NeuN, MAP2, or cell type-specific markers (TH for dopaminergic neurons)

• Astrocytes: GFAP

• Microglia: Iba1

• Oligodendrocytes: MBP or Olig2

• Neurotransmitter systems: markers for glutamatergic, GABAergic, cholinergic neurons

For biotin-conjugated OPRL1 antibodies, researchers must carefully select compatible detection systems for the second (or third) marker to avoid cross-reactivity. Streptavidin conjugates with spectrally distinct fluorophores offer flexibility in designing multiplex immunofluorescence protocols .

How can OPRL1 antibodies be leveraged in pain research and analgesic drug development?

OPRL1 plays a critical role in nociception and pain modulation, making it a valuable target for pain research and analgesic drug development. OPRL1 antibodies enable several key research applications:

Research has demonstrated that OPRL1 signaling has complex effects on nociception, with both pro- and anti-nociceptive actions depending on the anatomical context and specific pain modality. For example, studies have shown that pharmacological blockade of CXCR3 by (±)-NBI-74330 reduces neuropathic pain and enhances opioid effectiveness .

The biotin-conjugated OPRL1 antibody offers particular advantages for multiplexed imaging studies, allowing simultaneous visualization of OPRL1 with other pain-related receptors and signaling molecules. This approach is valuable for understanding how OPRL1 integrates with broader pain-processing networks in both physiological and pathological states .

What role do OPRL1 antibodies play in understanding neurodegenerative and psychiatric disorders?

OPRL1 antibodies are valuable tools for investigating the receptor's involvement in various neurological and psychiatric conditions:

Research indicates that OPRL1 has been found to play an essential role in cognition and to modulate inflammation and immune responses . Additionally, the NOP system has important roles in anxiety, depression, addiction, Parkinson's disease, and other conditions affecting cognition and emotional processing .

Biotin-conjugated OPRL1 antibodies are particularly valuable for studies requiring high sensitivity or multiplexed detection approaches. For example, in post-mortem brain tissue analysis, where antigen preservation may be suboptimal, the signal amplification provided by biotin-streptavidin systems can enhance detection sensitivity .

How are OPRL1 antibodies contributing to cancer research and potential therapeutic development?

Emerging research has revealed important roles for OPRL1 in cancer biology, opening new avenues for investigation:

Research has demonstrated that OPRL1 promotes autophagy through NF-κB signaling and is transcriptionally regulated by E2F1 in hepatocellular carcinoma (HCC) . This suggests potential roles in cancer cell survival and treatment response. Additionally, OPRL1 has been detected in gliomas tissue and cell lines like U-87 MG, indicating potential relevance in brain tumors .

For cancer research applications, biotin-conjugated OPRL1 antibodies offer advantages for tissue microarray screening and multiplex detection systems, enabling high-throughput analysis across multiple tumor samples and co-localization studies with cancer signaling markers .

What are the current limitations in OPRL1 antibody research and potential future developments?

Despite significant advances in OPRL1 antibody development and applications, several limitations and opportunities for improvement remain:

Future directions for OPRL1 antibody research include the development of more specific tools for detecting different receptor states (active vs. inactive, phosphorylated vs. non-phosphorylated) and the creation of nanobodies or other novel binding proteins with enhanced tissue penetration and reduced cross-reactivity .

The continued refinement of biotin-conjugated antibodies and compatible detection systems will further enhance the utility of these tools for multiplexed analysis, enabling more comprehensive understanding of OPRL1's integration within complex signaling networks in both physiological and pathological contexts.

How should researchers integrate OPRL1 antibody data with other research methodologies?

Maximizing the value of OPRL1 antibody-based research requires integration with complementary methodologies:

A multimodal approach that combines antibody-based detection of OPRL1 with functional and genetic approaches provides the most comprehensive understanding of this receptor's biology. For example, the integration of Western blot data showing OPRL1 expression changes with functional outcomes in nociception models has revealed important insights into the receptor's role in pain processing .