OPRL1 Antibody, FITC conjugated

What is OPRL1 and how does it relate to other opioid receptors?

OPRL1 (Opiate Receptor-Like 1) is a member of the G-protein coupled receptor 1 family and represents a distinct opioid receptor subtype. While traditionally grouped with classical opioid receptors (μ, δ, κ), OPRL1 has unique pharmacological properties and is also known as the nociceptin receptor, NOP receptor, ORL1, or orphanin FQ receptor. OPRL1 is structurally similar to other opioid receptors but responds preferentially to nociceptin/orphanin FQ rather than classical opioid ligands. The signaling via G proteins mediates inhibition of adenylate cyclase activity and calcium channel activity. Additionally, arrestins modulate signaling through G proteins and mediate the activation of alternative signaling pathways that lead to the activation of MAP kinases .

What is the cellular localization and functional significance of OPRL1?

OPRL1 is a multi-pass membrane protein primarily located in the cell membrane, but it can also be found in cytoplasmic vesicles following ligand binding. Functionally, ligand binding triggers receptor internalization into cytoplasmic vesicles, decreasing the receptor availability at the cell surface. This internalization process requires phosphorylation at Ser-363. OPRL1 can recycle back to the cell membrane after internalization. The receptor has been detected in blood leukocytes and plays roles in modulating nociception, anxiety, and various neurological processes .

How do OPRL1 expression patterns differ from other opioid receptors in human tissues?

Unlike μ-opioid receptors (OPRM1) that are expressed in specific nociceptive neuronal populations, OPRL1 shows a distinct expression pattern. While OPRM1 distinguishes two human nociceptive populations (OPRM1-positive and OPRM1-negative), OPRL1 has its own expression profile. In contrast, κ-opioid receptors (OPRK1) have been observed to be ubiquitously expressed in non-neuronal cells, mostly in subpopulations of satellite glial cells surrounding somatosensory neurons, rather than in neurons themselves . OPRL1 is encoded on human chromosome 20q13.33 and exhibits specific expression patterns that differ from classical opioid receptors, creating distinct opportunities for targeted research .

What are the key specifications of OPRL1 Antibody, FITC conjugated?

The OPRL1 Antibody, FITC conjugated (such as ABIN7161667) is a polyclonal antibody raised in rabbit that specifically targets the amino acid sequence 323-370 of human OPRL1. The antibody is directly conjugated to fluorescein isothiocyanate (FITC) fluorophore, enabling direct visualization in fluorescence-based applications. It demonstrates reactivity with human OPRL1 and is purified using Protein G with >95% purity. The immunogen used for antibody production is recombinant Human Nociceptin receptor protein (323-370AA), and the antibody isotype is IgG .

How should OPRL1 Antibody, FITC conjugated be stored for optimal stability?

For optimal stability and performance, the OPRL1 Antibody, FITC conjugated should be stored at either -20°C or -80°C upon receipt. It is important to avoid repeated freeze-thaw cycles as these can degrade the antibody and reduce its efficacy. The antibody is typically supplied in a liquid format with a buffer containing preservative (0.03% Proclin 300) and constituents (50% Glycerol, 0.01M PBS, pH 7.4) designed to maintain stability. When working with the antibody, aliquoting is recommended to minimize freeze-thaw cycles .

What is the difference between FITC-conjugated OPRL1 antibodies and other conjugates?

FITC-conjugated OPRL1 antibodies are specifically designed for fluorescence-based applications due to the direct attachment of the fluorescein isothiocyanate fluorophore to the antibody. This differs from other available conjugates such as HRP (horseradish peroxidase) which is optimized for ELISA and other enzymatic detection methods, or biotin conjugates which enable streptavidin-based detection systems. The FITC conjugation enables direct detection without the need for secondary antibodies in applications like flow cytometry, immunofluorescence microscopy, and live cell imaging. Different conjugates are suitable for different experimental approaches, allowing researchers to select the most appropriate tool for their specific research question .

What are the optimal applications for OPRL1 Antibody, FITC conjugated?

OPRL1 Antibody, FITC conjugated is specifically designed for direct immunofluorescence applications. The most suitable applications include:

• Flow cytometry: For quantitative analysis of OPRL1-expressing cells in suspension

• Live cell imaging: For real-time visualization of OPRL1 localization and trafficking

• Immunofluorescence microscopy: For detecting OPRL1 in fixed tissue or cell samples

• Receptor internalization studies: For tracking OPRL1 movement from membrane to cytoplasmic vesicles

The direct FITC conjugation eliminates the need for secondary antibodies, reducing background and simplifying experimental protocols. For optimal results, researchers should validate the antibody in their specific experimental system, as application suitability may vary depending on tissue type and experimental conditions .

How can OPRL1 Antibody, FITC conjugated be used to study receptor internalization?

OPRL1 receptor internalization is a critical process that occurs after ligand binding and requires phosphorylation at Ser-363. To study this process using FITC-conjugated OPRL1 antibody:

• Establish baseline receptor localization through membrane staining in live or fixed cells

• Stimulate cells with appropriate ligands (e.g., nociceptin/orphanin FQ)

• Monitor fluorescence signal redistribution from membrane to cytoplasmic vesicles using time-lapse microscopy

• Quantify internalization by measuring the ratio of membrane to cytoplasmic fluorescence over time

This approach can be complemented with inhibitors of receptor phosphorylation or endocytosis to dissect the molecular mechanisms involved. The FITC conjugation allows direct visualization without additional staining steps, enabling real-time tracking of receptor trafficking .

What protocol modifications are needed for flow cytometric analysis of OPRL1 using FITC-conjugated antibodies?

When performing flow cytometric analysis using OPRL1 Antibody, FITC conjugated:

• Cell preparation: Prepare single-cell suspensions and fix if necessary (4% paraformaldehyde, 10 minutes)

• Permeabilization: For intracellular detection, permeabilize with 0.1% Triton X-100 (omit for surface-only staining)

• Blocking: Block non-specific binding with 1-5% BSA or serum from the same species as the secondary antibody

• Primary antibody incubation: Dilute OPRL1 Antibody, FITC conjugated to appropriate concentration (typically 1-10 μg/ml) in blocking buffer and incubate for 30-60 minutes at room temperature

• Washing: Wash 3 times with PBS containing 0.1% BSA

• Analysis: Analyze samples using flow cytometer with appropriate filters for FITC (excitation ~495 nm, emission ~520 nm)

• Controls: Include an isotype control (FITC-conjugated rabbit IgG) to determine background fluorescence

This protocol can be optimized depending on the specific cell type and expression level of OPRL1 .

What are common issues when working with FITC-conjugated antibodies and how can they be resolved?

When working with OPRL1 Antibody, FITC conjugated, researchers may encounter several challenges:

For optimal results, validate the antibody using positive control samples known to express OPRL1 and determine the specificity by testing on tissues known to express OPRL1 positively and negatively .

How can researchers validate the specificity of OPRL1 Antibody, FITC conjugated?

Validating antibody specificity is crucial for reliable experimental results. For OPRL1 Antibody, FITC conjugated:

• Positive and negative controls: Test on tissues or cell lines known to express OPRL1 (e.g., blood leukocytes as positive control) and those that don't express OPRL1

• Peptide competition: Pre-incubate the antibody with the immunizing peptide (AA 323-370) to block specific binding

• siRNA knockdown: Reduce OPRL1 expression in cells using siRNA and confirm reduced antibody binding

• Western blot correlation: Verify that the size of detected bands matches the expected molecular weight (40.693 kDa)

• Multiple antibody validation: Compare staining patterns with different antibodies targeting different OPRL1 epitopes

• Co-localization studies: Confirm that staining patterns align with expected subcellular localization (membrane and cytoplasmic vesicles)

These validation steps ensure that the observed signal is specific to OPRL1 rather than due to non-specific binding or background fluorescence .

What considerations are important when designing co-labeling experiments with OPRL1 Antibody, FITC conjugated?

When designing co-labeling experiments:

• Spectral compatibility: Select additional fluorophores with minimal spectral overlap with FITC (avoid PE or other green fluorophores)

• Antibody species considerations: Choose primary antibodies raised in different host species to avoid cross-reactivity

• Epitope accessibility: Consider whether multiple antibodies might compete for closely located epitopes

• Sequential versus simultaneous staining: Determine whether antibodies should be applied simultaneously or sequentially

• Controls for cross-reactivity: Include single-stained controls to verify absence of bleed-through between channels

• Blocking optimization: Adjust blocking protocols to minimize background across all channels

• Fixation compatibility: Ensure fixation method preserves all target epitopes

For OPRL1 co-expression studies, consider co-staining with antibodies against other related receptors (OPRM1, OPRD1) or signaling partners using compatible fluorophores like Cy3, Cy5, or Alexa fluors to distinguish between different opioid receptor populations .

How can OPRL1 Antibody, FITC conjugated be used to investigate receptor heteromerization with other opioid receptors?

Investigating OPRL1 heteromerization with other opioid receptors requires sophisticated approaches:

• Proximity ligation assay (PLA): Use OPRL1 Antibody, FITC conjugated alongside antibodies against other opioid receptors (OPRM1, OPRD1, or OPRK1) with appropriate PLA probes to detect receptor pairs in close proximity

• FRET analysis: Combine FITC-conjugated OPRL1 antibody with acceptor fluorophore-labeled antibodies against other receptors to measure energy transfer indicating molecular proximity

• Co-immunoprecipitation followed by immunofluorescence: Pull down protein complexes and analyze co-precipitated receptors

• Single-molecule tracking: Use photoconvertible fluorescent proteins or quantum dots in combination with FITC-labeled antibodies to track receptor movement and interaction

• Super-resolution microscopy: Employ techniques like STORM or PALM to visualize nanoscale receptor clustering beyond the diffraction limit

These approaches can reveal how OPRL1 forms functional complexes with other opioid receptors, potentially explaining cross-talk between signaling pathways and unique pharmacological properties .

What experimental approaches can distinguish between different OPRL1-positive cell populations using FITC-conjugated antibodies?

To characterize different OPRL1-positive cell populations:

• Multi-parametric flow cytometry: Combine OPRL1 Antibody, FITC conjugated with antibodies against lineage-specific markers to identify distinct OPRL1+ subpopulations

• Single-cell RNA sequencing with protein detection: Correlate OPRL1 protein expression (detected via FITC) with transcriptomic profiles

• Spatial transcriptomics: Combine in situ hybridization for specific markers with OPRL1 immunofluorescence

• FACS sorting and functional assays: Sort OPRL1+ populations based on FITC signal intensity and perform functional characterization

• Spectral flow cytometry: Use advanced flow cytometry to distinguish subtle differences in OPRL1 expression levels across populations

Research indicates that different neuronal populations express distinct patterns of opioid receptors. For example, some populations express both OPRM1 and OPRL1, while others express OPRL1 alone or in combination with OPRD1. Understanding these expression patterns is crucial for developing targeted therapeutic approaches .

How can researchers quantitatively assess OPRL1 internalization kinetics using FITC-conjugated antibodies?

Quantitative assessment of OPRL1 internalization kinetics requires sophisticated imaging and analysis:

• Time-lapse confocal microscopy: Monitor FITC-labeled OPRL1 trafficking in real-time following ligand stimulation

• Fluorescence intensity ratio analysis: Measure the ratio of membrane to cytoplasmic fluorescence intensity over time

• Photo-bleaching approaches: Use FRAP (Fluorescence Recovery After Photobleaching) to measure mobility of receptors

• Pulse-chase experiments: Label surface receptors, induce internalization, and track remaining surface signal versus internalized signal

• Automated image analysis: Develop algorithms to quantify receptor clustering, internalization, and recycling

• Flow cytometry-based internalization assay: Compare surface versus total OPRL1 expression at different time points

Quantitative data can be presented as:

• Percent internalization versus time

• Internalization half-time (t½)

• Internalization rate constants

• Recycling efficiency

Since OPRL1 internalization requires phosphorylation at Ser-363, researchers can compare wild-type versus phosphorylation-deficient mutants to understand the molecular mechanisms governing receptor trafficking .