OR10J5 Antibody

What is OR10J5 and what is its molecular structure?

OR10J5 (Olfactory Receptor Family 10 Subfamily J Member 5) is a G-protein coupled receptor originally identified in the olfactory system but now known to have functional roles in diverse tissues. The receptor has a calculated molecular weight of 34,401 Da, though the observed molecular weight in laboratory applications is approximately 72 kDa, likely due to post-translational modifications and/or glycosylation patterns . OR10J5 belongs to the G-protein coupled receptor 1 family and contains the characteristic seven-transmembrane domain structure. The C-terminal region contains the sequence "CIDTTINEII NYGVSSFVIF VPIGLIFISY VLVISSILQI ASAEGRKKTF" which is often targeted for antibody production .

What known ligands activate OR10J5?

OR10J5 has been confirmed to respond to multiple ligands, with the most well-characterized being:

• α-cedrene: A sesquiterpene constituent of cedarwood oil derived from Cupressus and Juniperus species

• Lyral: A synthetic odorant

Computational docking studies have shown that α-cedrene demonstrates higher binding affinity for OR10J5 than lyral, though both successfully activate the receptor and increase intracellular cAMP levels . These findings indicate that OR10J5 may recognize multiple structurally distinct ligands, which has important implications for understanding its physiological functions beyond olfaction.

What are the validated applications for OR10J5 antibodies?

Different commercially available OR10J5 antibodies have been validated for various applications:

For immunofluorescence applications, researchers have successfully used OR10J5 antibodies to visualize receptor expression in MCF7 cells, demonstrating specific staining that can be blocked with synthesized peptide, confirming specificity .

How should heterologous expression systems be designed for studying OR10J5?

When designing heterologous expression systems to study OR10J5 function:

• Use FLAG-tagged full-length constructs encoding OR10J5 for ease of detection

• Express in Hana3A cells, which have been validated for olfactory receptor expression

• Verify surface expression using multiple methods:

  • Western blotting with FLAG antibody

  • Confocal imaging to confirm membrane localization (green signal should localize to plasma membrane)

  • Flow cytometric analysis to quantify percentage of cells expressing the receptor (approximately 50% of Hana3A cells can be expected to show surface expression)

This multi-method verification approach ensures proper receptor trafficking and expression before functional studies .

How can researchers measure OR10J5 activation in experimental models?

To effectively measure OR10J5 activation, researchers should implement multiple complementary approaches:

• cAMP accumulation assays: Since OR10J5 couples to G proteins that activate adenylyl cyclase, measuring cAMP is a primary readout. Hana3A cells heterologously expressing OR10J5 show significant increases in cAMP production when stimulated with either α-cedrene or lyral compared to empty vector controls .

• Calcium imaging: OR10J5 activation can trigger intracellular calcium mobilization, particularly in endothelial cells where it has been demonstrated to stimulate migration through calcium-dependent AKT signal transduction .

• Downstream pathway analysis: Monitor PKA activation, as the OR10J5–cAMP–PKA pathway has been implicated in hepatic lipid metabolism regulation .

When designing these experiments, include appropriate controls including cells transfected with empty vectors and receptor-specific knockdown conditions to confirm specificity of observed responses.

What are effective approaches for OR10J5 loss-of-function studies?

RNA interference-mediated knockdown has been successfully employed to study OR10J5 function in human hepatocytes. This approach revealed that:

• Knockdown of OR10J5 leads to increased intracellular lipid accumulation

• OR10J5 knockdown results in upregulation of lipogenic genes

• Genes related to fatty acid oxidation are downregulated following OR10J5 knockdown

For effective knockdown validation, researchers should quantify:

• Reduction in OR10J5 mRNA levels by qPCR

• Protein reduction via Western blot or immunofluorescence

• Functional impairment through reduced cAMP production in response to known agonists

Alternative approaches may include CRISPR-Cas9 genome editing for complete receptor knockout, though this method is not specifically detailed in the provided search results.

How does OR10J5 regulate metabolic processes in hepatocytes?

OR10J5 plays a crucial role in hepatic lipid metabolism, with multiple lines of evidence demonstrating:

• α-Cedrene stimulation significantly reduces lipid contents in human hepatocytes

• This effect is mediated specifically through OR10J5, as demonstrated by receptor knockdown experiments

• The signaling pathway involves the OR10J5–cAMP–PKA cascade

• OR10J5 activation leads to reprogramming of metabolic signatures in hepatocytes

Mechanistically, when OR10J5 is activated by ligands such as α-cedrene or lyral, it triggers increased cAMP production, which activates PKA. This signaling cascade ultimately downregulates lipogenic gene expression while upregulating genes involved in fatty acid oxidation, resulting in decreased triglyceride accumulation in hepatocytes .

How can researchers reconcile differences between calculated and observed molecular weights of OR10J5?

The discrepancy between the calculated molecular weight (34,401 Da) and observed molecular weight (~72 kDa) of OR10J5 presents an interesting research challenge . To investigate this phenomenon:

• Examine post-translational modifications: Perform enzymatic deglycosylation using PNGase F or similar enzymes to determine if N-linked glycosylation contributes to the molecular weight difference

• Analyze protein complexes: Use non-denaturing gel electrophoresis to determine if OR10J5 forms stable dimers or associates with other proteins

• Perform mass spectrometry analysis: Compare the mass of the native protein with theoretical predictions to precisely identify modifications

• Site-directed mutagenesis: Systematically mutate potential modification sites and observe effects on apparent molecular weight

This molecular weight discrepancy is not uncommon for membrane proteins, particularly GPCRs, which often exhibit anomalous migration on SDS-PAGE due to their hydrophobic nature and post-translational modifications.

Beyond olfactory tissues, where else is OR10J5 functionally expressed?

OR10J5 demonstrates significant extranasal expression with distinct functional roles:

• Hepatocytes: Regulates lipid metabolism and triglyceride accumulation through cAMP-PKA signaling

• Endothelial cells: Functions as a key regulator of angiogenesis, stimulating migration of human umbilical vein endothelial cells by activating calcium-dependent AKT signaling pathways

• Skeletal muscle: The mouse ortholog of OR10J5 (MOR23) is necessary for proper skeletal muscle regeneration, with loss of function leading to increased myofiber branching associated with muscular dystrophy

• Reproductive system: MOR23 (mouse ortholog) is functionally expressed in spermatogenic cells and sperm, where its activation increases intracellular calcium and regulates sperm motility

This diversity of expression sites suggests OR10J5 may serve as a chemosensor in multiple physiological contexts beyond its classical role in olfaction.

How does OR10J5 compare to its mouse ortholog MOR23 in terms of function and expression?

OR10J5 and its mouse ortholog MOR23 share significant functional similarities:

• Ligand recognition: Both receptors recognize similar odorants, including lyral and α-cedrene, with α-cedrene showing better binding affinity than lyral for both receptors according to docking studies

• Signaling mechanism: Both trigger increases in cAMP levels when stimulated with their respective ligands

• Expression patterns: Both show expression beyond olfactory tissues, though there may be species-specific differences in expression levels across tissues

• Functional roles: While OR10J5 has been implicated in hepatic lipid metabolism and angiogenesis in humans, MOR23 has documented roles in sperm motility and skeletal muscle regeneration in mice

These shared properties make mouse models potentially valuable for studying OR10J5 function, though researchers should remain cautious about species-specific differences.

What are effective storage and handling protocols for OR10J5 antibodies?

For optimal antibody performance and longevity:

• Long-term storage: Store at -20°C for up to one year

• Working storage: For frequent use, store at 4°C for up to one month

• Avoid freeze-thaw cycles: Repeated freezing and thawing significantly reduces antibody activity

• Storage buffer: Most commercial preparations contain PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide for stability

When retrieving antibody from storage, allow it to equilibrate to room temperature before opening to prevent condensation that could introduce contamination or dilute the antibody solution.

How can researchers validate the specificity of OR10J5 antibodies?

To ensure reliable results, validate OR10J5 antibody specificity through:

• Blocking peptide experiments: Use the synthetic peptide immunogen to compete with the antibody binding. The search results demonstrate successful blocking in immunofluorescence assays of MCF7 cells

• Positive and negative controls: Include tissues or cells known to express or lack OR10J5

• Knockdown/knockout validation: Compare staining between wild-type samples and those with reduced OR10J5 expression

• Multiple antibody comparison: Use antibodies targeting different epitopes of OR10J5 to confirm consistent results

• Cross-species reactivity: If using in non-human samples, verify sequence homology in the targeted epitope region. For example, one commercial antibody shows predicted reactivity of 92% in cow, dog, guinea pig, horse, mouse, and rat, but 100% in human and rabbit

Can OR10J5 antibodies validated for human applications be used in other species?

According to the search results, some OR10J5 antibodies have demonstrated cross-reactivity with multiple species:

• Boster Bio's A17073 antibody has been validated for human, mouse, and rat samples

• ABIN2791818 antibody has predicted reactivity for cow (92%), dog (92%), guinea pig (92%), horse (92%), mouse (92%), rabbit (100%), and rat (92%)

A researcher specifically asked about using the A17073 antibody (validated for human tissue) on monkey tissues. While not specifically tested, the manufacturer indicated "there is a good chance of cross reactivity" due to sequence conservation .

When applying antibodies across species:

• Compare epitope sequences between species to predict likelihood of cross-reactivity

• Start with higher antibody concentrations than used for validated species

• Include positive controls from validated species alongside experimental samples

• Consider using multiple detection methods to confirm results

What methodological adaptations are needed when studying OR10J5 in non-human models?

When extending OR10J5 research to non-human models:

• Antibody validation: Perform Western blots in the new species to confirm specificity and determine optimal working dilutions

• Sequence analysis: Compare the amino acid sequence of the target epitope between humans and the model species to estimate cross-reactivity potential

• Functional assays: When studying functional responses, consider possible differences in downstream signaling pathways between species

• Expression patterns: Evaluate whether tissue-specific expression patterns of OR10J5 are conserved across species

• Ligand responses: Verify that ligands identified for human OR10J5 (such as α-cedrene and lyral) evoke similar responses in the orthologous receptor in other species

These methodological considerations ensure robust cross-species comparisons and minimize misinterpretation of experimental results.