OR2AG2/OR2AG1 Antibody

What are OR2AG1 and OR2AG2, and what is their biological significance?

OR2AG1 and OR2AG2 are members of the olfactory receptor family, which function as G-protein-coupled receptors (GPCRs) with seven transmembrane domains. These receptors bind to specific odor molecules in the environment and are crucial for the detection and discrimination of different smells. They initiate a neuronal response that triggers the perception of smell when interacting with odorant molecules in the nose . OR2AG1 and OR2AG2 are encoded by single coding-exon genes and share structural similarities with many neurotransmitter and hormone receptors. The olfactory receptor gene family represents the largest gene family in the human genome, with each receptor having specificity for certain odorant molecules .

What are the key technical specifications of commercially available OR2AG1/OR2AG2 antibodies?

The OR2AG1/OR2AG2 Antibody (e.g., PACO01219) is typically a polyclonal antibody raised in rabbits against synthesized peptides derived from the internal region of human olfactory receptor 2AG1/2. The antibody specifications include:

These antibodies are typically affinity-purified to ensure high specificity for the target proteins .

How should researchers optimize Western blot protocols when using OR2AG1/OR2AG2 antibodies?

For optimal Western blot results with OR2AG1/OR2AG2 antibodies, researchers should implement the following methodology:

• Sample preparation: Extract proteins from tissues or cells expressing olfactory receptors using a RIPA buffer supplemented with protease inhibitors.

• Protein separation: Load 20-40μg of protein per lane on a 10-12% SDS-PAGE gel, as olfactory receptors have a molecular weight of approximately 35 kDa.

• Transfer and blocking: Transfer proteins to a PVDF membrane at 100V for 1 hour, then block with 5% non-fat milk in TBST for 1 hour at room temperature.

• Antibody incubation: Dilute the OR2AG1/OR2AG2 antibody at 1:1000 in blocking solution and incubate overnight at 4°C. The recommended range is 1:500-1:2000, so optimization may be necessary depending on expression levels .

• Detection: Use an HRP-conjugated anti-rabbit secondary antibody (1:5000) and develop with an enhanced chemiluminescence system.

• Controls: Include both positive controls (nasal epithelial tissue extracts) and negative controls (tissues not expressing OR2AG1/OR2AG2).

The expected molecular weight of OR2AG1/OR2AG2 is approximately 35,270 Da, which should be confirmed during validation .

What methodological approaches can be used to study OR2AG1/OR2AG2 localization in tissue samples?

For localization studies of OR2AG1/OR2AG2 in tissue samples, researchers can employ multiple complementary approaches:

• Immunofluorescence microscopy:

  • Fix tissue sections or cells with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

  • Block with 5% normal goat serum

  • Incubate with OR2AG1/OR2AG2 antibody at 1:500 dilution (optimization within 1:200-1:1000 range may be required)

  • Detect with fluorophore-conjugated secondary antibodies

  • Counterstain nuclei with DAPI

• Immunohistochemistry:

  • Use paraffin-embedded or frozen sections

  • Perform antigen retrieval if necessary

  • Apply a similar protocol as immunofluorescence but use HRP-conjugated secondary antibodies and DAB for visualization

• Confocal microscopy for co-localization studies:

  • Co-stain with markers for cellular compartments (e.g., membrane markers)

  • Use appropriate filters to distinguish fluorophores

  • Analyze co-localization quantitatively using specialized software

• Super-resolution microscopy:

  • For detailed subcellular localization, techniques like STORM or PALM can provide nanometer-scale resolution

  • Requires special sample preparation and fluorophores with appropriate photoswitching properties

These approaches should be validated with proper controls, including competitive peptide blocking and comparison with known expression patterns in olfactory epithelium .

How do molecular dynamics simulations contribute to understanding OR2AG1/OR2AG2 ligand binding mechanisms?

Molecular dynamics (MD) simulations provide critical insights into OR2AG1/OR2AG2 ligand binding by:

• Revealing dynamic conformational changes: MD simulations capture the three-dimensional motions of the receptor proteins when interacting with odorant molecules, showing how the binding pocket accommodates ligands and how this induces structural changes in the receptor .

• Elucidating binding kinetics: While cryo-electron microscopy may face limitations in resolving all structural details of olfactory receptors, MD simulations complement this by modeling the kinetics of odorant binding, including association and dissociation rates, and energy landscapes of the binding process .

• Identifying key binding residues: Simulations can identify which amino acids in the binding pocket form crucial interactions with ligands. For example, similar to studies on OR51E2, researchers can examine whether OR2AG1/OR2AG2 forms polar interactions (hydrogen and ionic bonds) and/or non-specific hydrophobic interactions with their ligands .

• Predicting binding site volume constraints: MD simulations can predict the volume of the binding pocket (as demonstrated for OR51E2's 31 ų pocket) and how this constrains which odorants can bind. For OR2AG1, research has identified amyl butyrate as a ligand, and simulations could reveal why this particular molecule fits the binding pocket .

• Testing effects of mutations: By performing virtual mutations of key residues in the binding pocket and re-running simulations, researchers can predict how genetic variations might affect ligand binding and receptor activation .

The integration of AlphaFold2-predicted 3D protein structures with MD simulations has greatly enhanced these applications, accelerating the deciphering of molecular mechanisms in olfactory signaling .

What are the known ligands for OR2AG1/OR2AG2, and how can researchers identify novel ligands?

Currently, the known ligand for OR2AG1 is amyl butyrate . For identifying novel ligands of OR2AG1/OR2AG2, researchers can employ several methodologies:

• High-throughput screening approaches:

  • Calcium imaging assays using cells expressing the receptor

  • GPCR activity reporter systems (e.g., GloSensor cAMP assay)

  • Label-free dynamic mass redistribution (DMR) assays

• Computational methods:

  • Protein Chemistry Metric (PCM) models that utilize OR sequence similarity and physicochemical characteristics of ligands

  • Machine learning approaches that can predict OR-odorant pairs with success rates of approximately 58%

  • Molecular docking and virtual screening techniques, which have shown 70% success rates in identifying novel antagonists or agonists for other olfactory receptors

• Structure-based drug design:

  • Based on the binding pocket characteristics of OR2AG1/OR2AG2

  • Synthetic analogues of amyl butyrate with modified functional groups to test structure-activity relationships

• Deorphanization strategies:

  • Screening libraries of structurally diverse odorants against cells expressing OR2AG1/OR2AG2

  • Using supervised machine learning techniques that consider the surrounding 60 residues of the binding pocket to predict potential ligands

Researchers should validate computational predictions with functional assays, such as calcium mobilization or cAMP production in heterologous expression systems.

How can cryo-electron microscopy be applied to study OR2AG1/OR2AG2 structure-function relationships?

Cryo-electron microscopy (cryo-EM) represents a cutting-edge approach for investigating OR2AG1/OR2AG2 structure-function relationships, as demonstrated by recent breakthroughs with other olfactory receptors:

• Sample preparation for cryo-EM:

  • Express and purify OR2AG1/OR2AG2 in a suitable expression system (e.g., HEK293 cells)

  • Stabilize the receptor using nanodiscs or amphipols to maintain native conformation

  • For co-structural studies, saturate the receptor with high-affinity ligands like amyl butyrate (for OR2AG1)

  • Flash-freeze samples in liquid ethane to preserve structural integrity

• Structural determination methodology:

  • Collect thousands of micrographs containing receptor particles in different orientations

  • Process images using motion correction and contrast transfer function estimation

  • Perform particle picking, 2D classification, and 3D reconstruction

  • Refine the structure to achieve near-atomic resolution (3-4 Å)

• Structure-activity correlation:

  • Compare structures of apo (unbound) and ligand-bound states to identify conformational changes

  • Analyze the extracellular loops, particularly ECL3, which has been shown to undergo structural alterations upon odorant binding in other olfactory receptors

  • Map the binding pocket dimensions and chemical properties to understand ligand specificity

• Integration with other techniques:

  • Combine cryo-EM with molecular dynamics simulations to model receptor dynamics beyond static structures

  • Validate structural insights using site-directed mutagenesis and functional assays

  • Apply insights from similar studies, such as those revealing the OR51E2 structure, which showed a compact, enclosed binding pocket that effectively traps odorant molecules

This integrated approach can reveal how OR2AG1/OR2AG2 selectivity is determined by binding pocket volume and amino acid composition, similar to findings that OR51E2 exclusively binds short-chain fatty acids due to its limited 31 ų binding pocket .

What are the challenges in studying post-translational modifications of OR2AG1/OR2AG2, and how can they be addressed?

Studying post-translational modifications (PTMs) of OR2AG1/OR2AG2 presents significant challenges due to their membrane localization, low expression levels, and technical limitations. Here's a methodological approach to address these challenges:

• Identification of potential PTM sites:

  • In silico prediction of phosphorylation, glycosylation, palmitoylation, and ubiquitination sites using specialized software tools

  • Analysis of evolutionary conservation of predicted PTM sites across species as an indicator of functional significance

• Experimental verification methods:

  • Mass spectrometry-based approaches:

    • Immunoprecipitate OR2AG1/OR2AG2 using validated antibodies

    • Apply specialized extraction protocols for membrane proteins

    • Use enrichment strategies specific to the PTM of interest (e.g., phosphopeptide enrichment using TiO₂ or IMAC)

    • Employ targeted mass spectrometry (PRM or MRM) for increased sensitivity

  • Site-directed mutagenesis:

    • Generate mutants of predicted PTM sites (e.g., Ser/Thr to Ala for phosphorylation sites)

    • Assess functional consequences using calcium imaging or cAMP assays

    • Compare trafficking and localization of wild-type vs. mutant receptors

• Temporal dynamics analysis:

  • Pulse-chase experiments to study PTM acquisition and turnover

  • Stimulus-dependent changes in PTM patterns following exposure to odorants

  • Time-course analysis of receptor activation and desensitization in relation to PTM status

• Visualization techniques:

  • Use PTM-specific antibodies in combination with the OR2AG1/OR2AG2 antibody for co-localization studies

  • Apply proximity ligation assays to detect specific PTMs on the receptor in situ

  • Implement FRET-based biosensors to monitor PTM events in real-time

These methods require rigorous validation with appropriate controls, including the use of PTM-blocking agents and comparison with well-characterized GPCRs as reference standards.

What is the evidence linking OR2AG2 genetic variations to asthma pathophysiology?

Evidence connecting OR2AG2 genetic variations to asthma pathophysiology comes from several research approaches:

• Genetic association studies:

  • A specific genetic variant in OR2AG2, rs10839616 (NM_001004490.1:c.161 G > C), was found to belong to a risk haplotype that co-segregated with all affected members in a multi-generational family with asthma

  • This association was validated in a separate cohort of 141 pediatric asthma cases compared with 130 controls

  • The variant shows evidence of unadjusted association with asthma in population-level analysis

• Physiological rationale:

  • Olfactory receptors are expressed in airway tissues, similar to bitter taste receptors

  • These receptors play roles in mucociliary clearance when activated by strong odors, suggesting a protective function in respiratory health

  • Impaired olfactory receptor function may contribute to reduced clearance of irritants or allergens, potentially triggering asthma exacerbations

• Functional evidence:

  • Phenotypic testing using olfactory identification and threshold tests with 2-phenylethyl alcohol (PEA) demonstrated altered olfactory function in affected individuals carrying the risk variant

  • This suggests that genetic variations in OR2AG2 may affect its normal sensory function

• Biological pathway relevance:

  • The finding supports the hypothesis that defects in sensing and clearance mechanisms mediated by olfactory receptors may trigger exacerbations, which is commonly observed in asthmatics exposed to strong or volatile odors

  • Pathway-level relevance to asthma biology has been tested in model systems and unrelated human lung samples

These findings suggest that OR2AG2 and potentially other olfactory receptors may contribute to asthma pathophysiology, opening new avenues for understanding asthma triggers and potentially developing novel therapeutic approaches.

How can researchers design functional studies to investigate OR2AG1/OR2AG2 signaling in airway epithelial cells?

To investigate OR2AG1/OR2AG2 signaling in airway epithelial cells, researchers should implement the following comprehensive methodological approach:

• Cell model selection and validation:

  • Primary human bronchial epithelial cells (HBECs) cultured at air-liquid interface

  • Immortalized airway epithelial cell lines (e.g., BEAS-2B, 16HBE14o-)

  • Confirmation of endogenous OR2AG1/OR2AG2 expression using RT-qPCR, Western blot with validated antibodies , and immunofluorescence

• Receptor activation studies:

  • Calcium flux assays using fluorescent indicators (Fluo-4 AM) to measure intracellular Ca²⁺ changes upon stimulation with known ligands (e.g., amyl butyrate for OR2AG1)

  • cAMP assays to measure G protein-coupled signaling responses

  • Real-time monitoring of cellular responses using biosensors for second messengers

  • Dose-response curves with varying concentrations of odorant molecules to determine EC₅₀ values

• Downstream signaling pathway characterization:

  • Phosphoprotein analysis of key signaling nodes (ERK, p38, JNK) using phospho-specific antibodies

  • Transcriptional profiling using RNA-seq to identify genes regulated by receptor activation

  • Pharmacological inhibitors to dissect specific pathway contributions

  • siRNA knockdown or CRISPR-Cas9 editing to confirm specificity of receptor-mediated effects

• Functional outcome assessment:

  • Mucociliary clearance measurements using micro-OCT imaging or fluorescent bead tracking

  • Ciliary beat frequency analysis using high-speed video microscopy

  • Mucus secretion quantification using ELISA for mucins

  • Airway surface liquid height measurements

• Integration with disease models:

  • Comparison of receptor function in cells from healthy donors versus asthmatic patients

  • Introduction of disease-associated genetic variants (e.g., rs10839616) using CRISPR-Cas9 knock-in approaches

  • Exposure to asthma-relevant stimuli (allergens, inflammatory cytokines) to assess receptor modulation

• Advanced three-dimensional models:

  • Airway organoids to study OR2AG1/OR2AG2 function in a more physiologically relevant context

  • Co-culture systems incorporating immune cells to investigate neuroimmune interactions

These approaches should include appropriate controls, including cells with receptor knockdown/knockout and stimulation with non-cognate ligands to confirm specificity of the observed responses.

What are common sources of non-specific binding when using OR2AG1/OR2AG2 antibodies, and how can researchers minimize them?

When working with OR2AG1/OR2AG2 antibodies, researchers may encounter non-specific binding issues that can compromise experimental results. Here are the common sources and methodological solutions:

• Cross-reactivity with other olfactory receptors:

  • The high sequence homology among the large family of olfactory receptors (ORs) can lead to antibody cross-reactivity

  • Solution: Perform pre-absorption controls by incubating the antibody with the immunizing peptide before application

  • Validate specificity using cells overexpressing OR2AG1/OR2AG2 versus other closely related ORs

  • Consider using knockout/knockdown controls where possible

• Membrane protein aggregation:

  • ORs are hydrophobic membrane proteins that can form aggregates during sample preparation

  • Solution: Optimize sample preparation by using appropriate detergents (e.g., DDM, CHAPS) at optimal concentrations

  • Ensure complete solubilization by adjusting incubation time and temperature

  • Include reducing agents (e.g., DTT) to prevent disulfide-mediated aggregation

• Inadequate blocking:

  • Insufficient blocking can lead to high background signal

  • Solution: Optimize blocking conditions by testing different blocking agents (BSA, non-fat milk, normal serum)

  • Extend blocking time to 2 hours at room temperature or overnight at 4°C

  • Include 0.1-0.3% Triton X-100 or Tween-20 in washing buffers to reduce hydrophobic interactions

• Secondary antibody issues:

  • Non-specific binding of secondary antibodies can contribute to background

  • Solution: Use highly cross-adsorbed secondary antibodies specific to the host species of the primary antibody

  • Reduce secondary antibody concentration

  • Include serum from the secondary antibody host species in the blocking buffer

• Endogenous peroxidase or phosphatase activity:

  • Can cause false positive signals in immunohistochemistry

  • Solution: Include a peroxidase/phosphatase quenching step (e.g., 3% H₂O₂ treatment)

  • Use appropriate enzyme inhibitors in the detection system

• Optimization strategy:

  • Perform a systematic titration of antibody concentrations (starting with the recommended 1:500-1:2000 for WB and 1:200-1:1000 for IF)

  • Include both positive controls (nasal epithelium tissue) and negative controls (tissues not expressing ORs)

  • Consider using monoclonal antibodies if available for higher specificity

These methodological adjustments should significantly reduce non-specific binding and improve the signal-to-noise ratio in experiments utilizing OR2AG1/OR2AG2 antibodies.

What approaches can resolve data inconsistencies when studying OR2AG1/OR2AG2 expression across different tissue types?

When researchers encounter inconsistent data regarding OR2AG1/OR2AG2 expression across different tissue types, the following methodological approach can help resolve discrepancies: