OPRK1 Antibody

What is OPRK1 and where is it expressed?

OPRK1 (Opioid Receptor, kappa 1) is a G protein-coupled receptor belonging to the opioid receptor family, which includes mu (μ), delta (δ), kappa (κ), and nociceptin/orphanin FQ (N/OFQ) receptors . The receptor functions in binding endogenous opiates such as endorphins, endomorphins, and enkephalins, as well as synthetic opioid compounds, resulting in inhibition of adenylate cyclase activity and neurotransmitter release . OPRK1 plays crucial roles in pain perception and mediating the hypolocomotor, analgesic, and aversive actions of opioids .

Expression of OPRK1 has been detected in multiple tissues and cell types, with significant expression in neural tissues. Immunohistochemical analysis has revealed OPRK1 expression in rat piriform cortex, appearing in both cells and processes . The receptor has also been detected in mouse and rat brain, rat testis, human stomach, mouse kidney, and human neuroblastoma (SH-SY5Y) cells . Recent research has additionally identified OPRK1 expression in prostate cancer tissues, with upregulation observed following androgen deprivation therapy or castration-resistant prostate cancer progression .

Validation of OPRK1 antibodies is essential to ensure experimental reliability. Several validation approaches are recommended:

Blocking peptide controls are crucial for confirming antibody specificity. For example, preincubation of Anti-κ-Opioid Receptor (OPRK1) antibody with κ-Opioid Receptor/OPRK1 blocking peptide can suppress staining in immunohistochemistry, confirming specificity . Similarly, in Western blot analysis, comparing samples with and without blocking peptide preincubation helps identify specific bands versus non-specific binding .

Multiple detection methods should be employed to cross-validate findings. If an antibody produces consistent results across Western blot, immunohistochemistry, and immunofluorescence applications in tissues known to express OPRK1, confidence in specificity increases .

Additionally, researchers should be aware that the observed molecular weight of OPRK1 may not always match theoretical predictions. For instance, while the calculated molecular weight is 33 kDa/42 kDa, the observed band in Western blot may appear at approximately 40 kDa due to post-translational modifications and protein processing .

What applications are OPRK1 antibodies suitable for?

OPRK1 antibodies can be utilized in various research applications depending on their specific properties:

Western Blotting (WB): Most OPRK1 antibodies are suitable for WB applications with recommended dilutions typically between 1:500-1:2000 . This technique enables detection of the receptor in tissue lysates and cellular extracts, with confirmed detection in mouse brain, rat brain, and human neuroblastoma cells .

Immunohistochemistry (IHC): OPRK1 antibodies have been successfully used for IHC in various tissues including rat piriform cortex, rat testis, human stomach, and mouse kidney . Optimal dilutions for IHC typically range from 1:50-1:200 .

Immunofluorescence (IF): Several OPRK1 antibodies are validated for IF applications, allowing for visualization of receptor localization in fixed tissue sections and cultured cells. This approach has successfully demonstrated OPRK1 immunoreactivity in cells and processes of the rat piriform cortex .

Flow Cytometry: Cell surface detection of OPRK1 can be achieved using antibodies targeting extracellular epitopes, as demonstrated in mouse J774 macrophage cells .

ELISA: Several OPRK1 antibodies are suitable for enzyme-linked immunosorbent assays, facilitating quantitative analysis of receptor expression .

How can OPRK1 antibodies be used to study receptor trafficking and internalization?

Investigating OPRK1 trafficking and internalization requires specific methodological approaches using antibodies targeting different epitopes. For receptors like OPRK1, internalization studies typically employ antibodies targeting extracellular domains (such as the N-terminus) that can label the receptor at the cell surface prior to stimulation with agonists .

To study receptor trafficking dynamics, researchers should consider dual-labeling approaches where cell surface receptors are labeled with one fluorophore before stimulation, while total cellular receptor pools are labeled with a different fluorophore after permeabilization. This allows discrimination between internalized and newly synthesized receptors. Antibodies targeting the extracellular domain of OPRK1, such as those recognizing amino acids 39-55 in the N-terminus, are particularly suitable for such studies .

Time-course experiments utilizing confocal microscopy and OPRK1 antibodies can reveal the kinetics of receptor internalization, recycling, and degradation following agonist stimulation. For quantitative analysis, flow cytometry with antibodies specific to extracellular epitopes can measure changes in cell surface receptor populations over time, as has been demonstrated with OPRK1 in J774 macrophage cells .

What is the role of OPRK1 in prostate cancer progression and how can antibodies help investigate this?

Recent research has identified OPRK1 as a potential key player in castration-resistant prostate cancer (CRPC) progression. Integrative genomic analysis using a patient-derived xenograft model revealed that OPRK1 harbors androgen receptor binding sites (ARBS) and is upregulated upon androgen deprivation . This suggests OPRK1 involvement in post-castration survival and cellular adaptation processes leading to castration resistance.

OPRK1 antibodies can be instrumental in investigating this phenomenon through several approaches:

Immunohistochemical analysis with OPRK1 antibodies has shown that expression is upregulated in human prostate cancer tissues after preoperative androgen derivation or CRPC progression . This makes OPRK1 antibodies valuable tools for monitoring receptor expression changes during disease progression and treatment response.

For functional studies, researchers can employ OPRK1 antibodies in combination with knockdown/knockout models to validate loss-of-function effects. Studies have demonstrated that OPRK1 loss of function retards the acquisition of castration resistance and inhibits castration-resistant growth of prostate cancer both in vitro and in vivo .

Co-immunoprecipitation experiments utilizing OPRK1 antibodies can help identify protein interaction partners that might contribute to castration resistance, providing insights into downstream signaling mechanisms. Western blot analysis with phospho-specific OPRK1 antibodies can further elucidate receptor activation status during disease progression .

How can phospho-specific OPRK1 antibodies be used to investigate receptor regulation?

Phosphorylation is a critical post-translational modification that regulates GPCR function, including receptor desensitization, internalization, and signaling pathway selection. Phospho-specific antibodies targeting OPRK1, such as those recognizing phosphorylated Ser369, provide valuable tools for investigating these regulatory mechanisms .

Methodologically, researchers can employ phospho-specific OPRK1 antibodies in Western blot analysis to monitor changes in receptor phosphorylation status following agonist stimulation or under different experimental conditions. This approach requires careful sample preparation to preserve phosphorylation status, including the use of phosphatase inhibitors during tissue or cell lysis .

Temporal dynamics of OPRK1 phosphorylation can be assessed through time-course experiments, where samples are collected at various intervals after agonist exposure. For spatial resolution, immunofluorescence with phospho-specific antibodies can reveal subcellular localization of phosphorylated receptors, potentially identifying signaling compartments within the cell .

To understand the functional consequences of OPRK1 phosphorylation, researchers can correlate phosphorylation levels (detected with phospho-specific antibodies) with downstream signaling events or physiological responses. This approach has been successfully applied to mouse and rat models, where phospho-Ser369 OPRK1 antibodies have demonstrated utility in Western blot, ELISA, and IHC applications .

What are the optimal conditions for Western blot analysis of OPRK1?

Western blot analysis of OPRK1 requires careful optimization to ensure specific detection and accurate interpretation. Based on available research, the following methodological approaches are recommended:

Sample preparation is critical, as OPRK1 is a membrane protein that may form aggregates during processing. Tissue or cell lysates should be prepared in buffers containing appropriate detergents (such as 1% Triton X-100 or 0.5% SDS) to solubilize membrane proteins effectively. Additionally, inclusion of protease inhibitors prevents protein degradation during processing .

For protein separation, 10-12% polyacrylamide gels are typically suitable for resolving OPRK1. It's essential to note that while the calculated molecular weight of OPRK1 is 33-42 kDa, the observed band often appears at approximately 40 kDa. This discrepancy may be attributed to post-translational modifications affecting protein mobility .

Including appropriate positive controls (tissues or cells known to express OPRK1, such as brain tissues) and negative controls (antibody preincubated with blocking peptide) is essential for validating specificity .

What considerations are important for immunohistochemical detection of OPRK1?

Successful immunohistochemical detection of OPRK1 requires attention to several technical aspects:

Fixation method significantly impacts epitope accessibility and antibody binding. For OPRK1 detection in brain tissues, perfusion fixation with 4% paraformaldehyde has proven effective, as demonstrated in studies of rat piriform cortex . For other tissues, such as testis, stomach, and kidney, standard formalin fixation followed by paraffin embedding may be suitable, though antigen retrieval steps are often necessary .

Antibody dilution must be optimized based on the specific tissue and detection system. For immunofluorescence in rat brain sections, a dilution of 1:300 has been successful with Anti-κ-Opioid Receptor (OPRK1) extracellular antibody . For IHC applications in other tissues, dilutions ranging from 1:50-1:200 are typically recommended, though optimal conditions should be determined empirically .

Detection systems should be selected based on the desired sensitivity and visualization method. For fluorescence detection, secondary antibodies conjugated to fluorophores (such as AlexaFluor-488) provide excellent sensitivity and specificity, as demonstrated in rat piriform cortex staining . For chromogenic detection, systems based on horseradish peroxidase or alkaline phosphatase can be employed.

Validation of specificity is crucial and can be achieved by including control sections treated with antibody preincubated with the corresponding blocking peptide. This approach has effectively demonstrated specificity in rat brain sections, where preincubation with κ-Opioid Receptor/OPRK1 blocking peptide suppressed staining .

How should researchers interpret discrepancies in OPRK1 molecular weight?

Researchers frequently encounter discrepancies between the calculated and observed molecular weights of OPRK1 in Western blot analyses. The calculated molecular weight is reported as 33 kDa/42 kDa, while the observed band typically appears at approximately 40 kDa . These discrepancies require careful interpretation:

Post-translational modifications significantly impact protein mobility in SDS-PAGE. OPRK1, like other GPCRs, undergoes various modifications including glycosylation, phosphorylation, and ubiquitination, which can alter its apparent molecular weight . Phosphorylation of sites such as Ser369 may contribute to altered migration patterns .

Alternative splicing of the OPRK1 gene results in different isoforms with varying molecular weights. Research has identified alternatively spliced transcript variants encoding different isoforms, which may explain some of the observed molecular weight heterogeneity .

Translational readthrough mechanisms have been reported for OPRK1, resulting in C-terminally extended isoforms through the use of alternative in-frame translation termination sites. This can produce protein variants with increased molecular weights compared to the canonical form .

To address these challenges, researchers should employ multiple approaches to confirm OPRK1 identity, including antibodies targeting different epitopes, blocking peptide controls, and comparison with positive control samples known to express OPRK1 .

How can researchers address non-specific binding when using OPRK1 antibodies?

Non-specific binding is a common challenge when working with OPRK1 antibodies, particularly in applications like Western blotting and immunohistochemistry. Several strategies can help minimize this issue:

Blocking optimization is crucial for reducing background signal. For Western blotting, extended blocking (1-2 hours at room temperature or overnight at 4°C) with 5% non-fat dry milk or 3-5% BSA in TBST (Tris-buffered saline with 0.1% Tween-20) can significantly reduce non-specific binding. For immunohistochemistry, including 5-10% serum from the species in which the secondary antibody was raised helps minimize background .

Antibody dilution optimization through titration experiments can identify the optimal concentration that maximizes specific signal while minimizing background. Starting with manufacturer recommendations (e.g., 1:500-1:2000 for Western blot or 1:50-1:200 for IHC) and testing a range of dilutions is a systematic approach to optimization .

Validation using blocking peptides provides a powerful control for distinguishing specific from non-specific signals. As demonstrated in Western blot analysis of mouse brain, rat brain, and human neuroblastoma cell lysates, comparison of results with and without antibody preincubation with a blocking peptide can clearly identify specific bands .

Multiple antibody verification, using antibodies targeting different epitopes of OPRK1, can help confirm specific signals. If multiple antibodies produce consistent results across different applications and tissues, confidence in specificity increases .

What approaches can be used to detect low levels of OPRK1 expression?

Detecting low abundance receptors like OPRK1 in certain tissues or experimental conditions requires specialized approaches:

Signal amplification techniques can significantly enhance detection sensitivity. For immunohistochemistry, tyramide signal amplification (TSA) can amplify signal 10-100 fold compared to conventional methods. For Western blotting, enhanced chemiluminescence (ECL) substrates with extended exposure times may improve detection of weak signals .

Sample enrichment through subcellular fractionation to isolate membrane proteins can concentrate OPRK1, improving detection in Western blotting. Similarly, immunoprecipitation using OPRK1 antibodies prior to Western blotting can enrich the target protein from complex samples .

Optimized extraction methods are essential, particularly for membrane proteins like OPRK1. Using specific lysis buffers designed for membrane proteins (containing appropriate detergents and chaotropic agents) can improve solubilization and recovery. For tissues with known low expression, increasing the amount of starting material and reducing the final volume can effectively concentrate the protein .

Enhanced detection systems, such as highly sensitive fluorophores for immunofluorescence or high-sensitivity ECL substrates for Western blotting, can significantly improve detection of low abundance targets. For flow cytometry, using brighter fluorophores and multi-layer detection systems (biotin-streptavidin) can enhance signal intensity .

How can researchers use OPRK1 antibodies to investigate receptor heteromerization?

OPRK1, like other opioid receptors, can form heteromeric complexes with other GPCRs, influencing signaling outcomes and pharmacological responses. Investigating these interactions requires specialized approaches using OPRK1 antibodies:

Proximity ligation assays (PLA) offer high sensitivity for detecting protein-protein interactions in situ. This technique uses pairs of antibodies targeting different receptors (e.g., OPRK1 and another GPCR) coupled with oligonucleotide probes. If the proteins are in close proximity (typically <40 nm), the probes can be ligated and amplified, generating a fluorescent signal at interaction sites.

Co-immunoprecipitation experiments using OPRK1 antibodies can pull down receptor complexes from tissue or cell lysates. Subsequent Western blotting with antibodies against potential interaction partners can identify heteromeric associations. For this application, antibodies targeting specific domains of OPRK1, such as the N-terminal or C-terminal regions, may be particularly useful .

Fluorescence resonance energy transfer (FRET) microscopy combined with immunofluorescence using OPRK1 antibodies can visualize receptor interactions in fixed cells or tissues. This approach requires antibodies labeled with appropriate donor and acceptor fluorophores, and careful controls to distinguish specific FRET signals from background.

For these advanced applications, antibody validation is particularly critical. Researchers should verify that the selected antibodies do not interfere with potential interaction domains and maintain specificity under the experimental conditions used for heteromerization studies .