OR6A2 Antibody

What is OR6A2 and what experimental systems can validate its expression?

OR6A2 is a protein encoded by the OR6A2 gene in humans and functions as a Class II (tetrapod-specific) olfactory receptor and rhodopsin-like receptor . It belongs to the large family of G-protein-coupled receptors (GPCRs) that share a 7-transmembrane domain structure with neurotransmitter and hormone receptors .

For validating OR6A2 expression:

• mRNA detection: RT-PCR or RNA sequencing can detect OR6A2 mRNA in tissues of interest

• Protein detection: Western blot using validated anti-OR6A2 antibodies shows bands at approximately 36-38 kDa (calculated) though observed molecular weight may be higher (~72 kDa) due to post-translational modifications

• Cellular localization: Immunofluorescence microscopy using anti-OR6A2 antibodies can determine cellular and subcellular distribution

Which applications are commercial OR6A2 antibodies validated for?

Current commercially available OR6A2 antibodies have been validated for specific research applications:

Note that most commercial antibodies are specifically labeled for research use only and not for diagnostic applications .

What are the optimal storage conditions for OR6A2 antibodies?

For maximum stability and activity retention of OR6A2 antibodies:

• Long-term storage: -20°C for up to one year in appropriate buffer conditions

• Short-term/frequent use: 4°C for up to one month

• Buffer composition: Typically supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• Avoid: Repeated freeze-thaw cycles as they can damage antibody structure and function

How does OR6A2/Olfr2 contribute to atherosclerosis pathogenesis and what experimental models validate this relationship?

Recent studies have revealed a significant role for OR6A2 (human ortholog of mouse Olfr2) in atherosclerosis through several mechanisms:

• Ligand detection: OR6A2/Olfr2 detects octanal, a product of lipid peroxidation found in human and mouse plasma at concentrations sufficient to activate the receptor

• Inflammasome activation: Upon octanal detection, OR6A2/Olfr2 activates the NLRP3 inflammasome in macrophages, leading to IL-1β and IL-1α secretion

• Disease progression: Genetic targeting of Olfr2 in mice significantly reduced atherosclerotic plaques, while boosting octanal levels exacerbated atherosclerosis

Experimental validation models:

• Olfr2-/- mice generated using CRISPR-Cas9 showed ~40% reduction in atherosclerotic lesion size

• Bone marrow transplantation from Olfr2-/- mice to atherosclerosis-prone recipients resulted in significantly smaller atherosclerotic lesions

• Human carotid endarterectomy samples from the BiKE dataset show increased OR6A2 expression correlating with plaque macrophage content

What is the relationship between OR6A2 expression and macrophage populations?

OR6A2/Olfr2 expression varies significantly across different macrophage populations:

• Vascular macrophages: Express high levels of OR6A2/Olfr2, with expression increasing in response to Western diet challenge

• Cell type distribution: Approximately 80% of Olfr2-expressing cells in vascular tissue are CD68+ macrophages, with the remaining 20% being other cell types, including endothelial cells

• LPS responsiveness: Treatment with LPS (TLR4 agonist) significantly increases Olfr2 expression in vascular macrophages, with further enhancement in the presence of octanal

• Tissue specificity: Different tissue macrophages express distinct subsets of olfactory receptors, with vascular macrophages showing higher normalized OR expression compared to naïve intraperitoneal macrophages and lung macrophages

What signaling mechanisms are associated with OR6A2 activation and how can they be experimentally manipulated?

OR6A2/Olfr2 signaling involves several molecular components and pathways:

• Required signaling components: Human macrophages express OR6A2 along with signaling components including GNAL, CNGA1, CNGA3, CNGA4, CNGB1, RTP1, and ADCY3

• Calcium signaling: Octanal induces Ca2+ flux in macrophages in response to LPS+octanal stimulation, which can be blocked by the olfactory receptor inhibitor citral

• Inflammasome activation: OR6A2/Olfr2 activation leads to NLRP3 inflammasome activation and subsequent IL-1β and IL-1α secretion

Experimental manipulation approaches:

• Genetic knockdown: siRNA targeting OR6A2 significantly reduces OR6A2 mRNA expression and IL-1β secretion in response to octanal

• Pharmacological inhibition: Citral can block calcium flux in response to octanal

• Inhibition of downstream effectors: Targeting caspase-1 or GSDMD significantly reduces IL-1β secretion following OR6A2/Olfr2 activation

• CRISPR-Cas9 targeting: Guide RNAs designed to target the OR6A2 gene can be used for genetic knockout studies

What is the mechanism underlying OR6A2 genetic variation and coriander taste perception?

OR6A2 genetic variation has been identified as a likely cause for the polarized perception of coriander (cilantro) taste:

• Population differences: The proportion of people reporting dislike of coriander varies by ancestry: 21% for East Asians, 17% for Caucasians, 14% for those of African descent, 7% for South Asians, 4% for Hispanics, and 3% for Middle Eastern subjects

• Sensory perception: Those with certain OR6A2 variants associate coriander with an intensely unpleasant taste, described as a combination of soap and vomit, or similar to stinkbug odor

• Molecular basis: This perception difference is suggested to be due to differential detection of aldehyde chemicals present in coriander, which are also found in soap, various detergents, and stinkbugs

Research approaches to study this phenomenon:

• Genetic association studies comparing OR6A2 variants across populations with different coriander perception

• Functional studies using in vitro expression systems to characterize receptor response to coriander aldehydes

• Structural biology approaches to understand how genetic variations alter binding site configuration

What are optimal strategies for using OR6A2 antibodies in complex tissue samples?

When working with OR6A2 antibodies in complex tissue samples, researchers should consider:

Optimization strategies:

• Antibody selection: Choose antibodies with validated reactivity to your species of interest. Current commercial antibodies react with human, mouse, and rat OR6A2

• Dilution optimization: For Western blot, start with 1:500-1:2000 dilution and adjust based on signal strength and background

• Blocking: Use the immunizing peptide as a negative control to confirm specificity, as demonstrated in validation studies

• Cross-reactivity assessment: While some antibodies may cross-react with non-validated species like primates, this should be experimentally confirmed

• Special formulations: For conjugation experiments, request BSA-free and azide-free formulations with appropriate cryoprotectants like trehalose or glycerol

Tissue-specific considerations:

• For brain tissue, preliminary testing is recommended as specific validation in neural tissues may not be available for all commercial antibodies

• For vascular tissue, consider co-staining with macrophage markers like CD68 to identify OR6A2-expressing macrophages

How can OR6A2 inhibition be leveraged as a therapeutic strategy for atherosclerosis?

Based on recent research, inhibiting OR6A2 presents a promising strategy for atherosclerosis treatment:

• Mechanism of action: Inhibiting OR6A2 would potentially reduce octanal-induced inflammasome activation in macrophages, leading to decreased IL-1β and IL-1α production

• Experimental evidence: Genetic targeting of Olfr2 (mouse ortholog) significantly reduced atherosclerotic plaque formation in mouse models

• Translational potential: Human carotid plaques show increased OR6A2 expression correlating with macrophage content, suggesting relevance to human disease

Potential therapeutic approaches:

• Development of small molecule antagonists targeting OR6A2

• Targeting receptor trafficking components like RTP1/RTP2 that are required for OR6A2 function

• Inhibiting downstream signaling pathways activated by OR6A2

• Gene therapy approaches using CRISPR-Cas9 to reduce OR6A2 expression in vascular macrophages

Researchers have proposed that "drug-like small molecules targeting OR6A2 and possibly other OLFRs may constitute novel therapeutic targets for the treatment, prevention, and reversal of atherosclerosis" .

What are the critical controls when performing OR6A2 antibody-based experiments?

Proper experimental controls are essential for reliable OR6A2 antibody-based research:

Positive controls:

• Cell lines known to express OR6A2 (e.g., Jurkat cells, HeLa cells)

• Recombinant OR6A2 protein (if available)

• Tissues with confirmed OR6A2 expression (e.g., vascular tissue with macrophage infiltration)

Negative controls:

• Blocking with immunizing peptide (demonstrated in validation images for several commercial antibodies)

• OR6A2 knockout or knockdown samples

• Secondary antibody only controls

• Isotype controls: Rabbit IgG controls are available for rabbit polyclonal anti-OR6A2 antibodies

Methodology validation:

• For Western blot applications, the observed molecular weight (~72 kDa) may differ from the calculated weight (36 kDa) due to post-translational modifications

• For immunofluorescence, co-staining with cell-type specific markers (e.g., CD68 for macrophages) can confirm cell-type specific expression

How can researchers quantify OR6A2 expression changes in experimental conditions?

Accurate quantification of OR6A2 expression changes requires appropriate methodologies:

mRNA quantification:

• RT-qPCR with validated primers specific to OR6A2

• RNA sequencing with appropriate normalization strategies

• Compare expression to housekeeping genes like GAPDH, as used in studies analyzing OR expression across macrophage populations

Protein quantification:

• Western blot with densitometric analysis

• Flow cytometry for cellular expression levels

• ELISA for quantification in lysates or supernatants

• Mass spectrometry-based proteomics

Normalization strategies:

• For comparing OR6A2 expression across different tissues or conditions, researchers have used the ranking strategy: (X-Xmin)/(Xmax-Xmin) with "X" being the respective OR expression level and "Xmin" the minimum level of OR expression per dataset

• For Western blot, normalization to housekeeping proteins like β-actin or GAPDH