OR9G1 Antibody, HRP conjugated

What is the OR9G1 protein and why is it significant in research?

OR9G1 (olfactory receptor family 9 subfamily G member 1) is a protein-coding gene that belongs to the large family of G-protein-coupled receptors (GPCRs). Also known as OR9G5, this receptor is part of the olfactory receptor family that typically interacts with odorant molecules in the nose to initiate neuronal responses triggering smell perception . OR9G1 shares the characteristic 7-transmembrane domain structure common to many neurotransmitter and hormone receptors . The significance of OR9G1 extends beyond olfactory functions, as research has revealed that olfactory receptors are expressed in non-chemosensory organs, including neurons of the central nervous system (CNS) in both humans and mice . This unexpected expression pattern suggests potentially novel functions in the nervous system, making OR9G1 an intriguing target for neuroscience research.

What are the key specifications of commercially available OR9G1 Antibody, HRP conjugated?

The commercially available OR9G1 Antibody, HRP conjugated is a polyclonal antibody raised in rabbits against recombinant Human Olfactory receptor 9G1 protein, specifically targeting amino acids 290-304 . This antibody is directly conjugated with horseradish peroxidase (HRP), which facilitates direct detection without requiring secondary antibodies. It has been validated for ELISA applications and shows reactivity with human species . The antibody is typically supplied in liquid form containing preservative (0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) . It has undergone Protein G purification to achieve >95% purity and belongs to the IgG isotype . For optimal performance, researchers should store the antibody at -20°C or -80°C and avoid repeated freeze-thaw cycles .

How does HRP conjugation benefit antibody applications in OR9G1 research?

HRP conjugation to OR9G1 antibodies provides several significant benefits for research applications. The direct conjugation eliminates the need for secondary antibody incubation steps in detection protocols, which streamlines experimental procedures and reduces background noise. In immunoassays such as ELISA, the HRP enzyme catalyzes colorimetric, chemiluminescent, or fluorescent reactions when exposed to appropriate substrates, allowing for sensitive detection of OR9G1 protein .

The enhanced sensitivity is particularly valuable when studying olfactory receptors like OR9G1 in the central nervous system, where expression levels may be lower than in the olfactory epithelium . HRP-conjugated antibodies have demonstrated superior dilution capabilities compared to unconjugated antibodies, with studies showing that optimized HRP-conjugation methods can enable antibody dilutions up to 1:5000 while maintaining signal, compared to conventional methods that may only work at dilutions as low as 1:25 . This improved sensitivity is crucial for detecting low-abundance proteins like OR9G1 in non-olfactory tissues.

How can OR9G1 expression patterns in neurodegenerative diseases inform experimental design?

Research has revealed that olfactory receptor gene expression, including OR family members, is altered in several neurodegenerative conditions such as Parkinson's disease (PD), Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and sporadic Creutzfeldt-Jakob disease (sCJD) . These changes exhibit disease-, region-, and subtype-specific patterns. For example, in Alzheimer's disease models using APP/PS1 transgenic mice, significant changes in olfactory receptor signaling pathways have been observed, with Olfr110 mRNA increasing from 3 months of age onwards .

When designing experiments to investigate OR9G1 in the context of neurodegenerative diseases, researchers should:

• Consider region-specific analyses, as expression changes may vary between brain regions.

• Include age-matched controls, as expression patterns change with disease progression.

• Examine multiple neurodegeneration markers simultaneously with OR9G1 to establish correlations.

• Design longitudinal studies when using animal models to capture disease progression effects.

• Include analyses of downstream signaling molecules like adenylyl cyclase 3 (AC3) and olfactory G protein α subunit (Gαolf), as these are functionally linked to olfactory receptors .

These considerations are essential for meaningful interpretation of OR9G1 antibody data in neurodegenerative disease research.

What are the methodological challenges in detecting OR9G1 in non-olfactory neural tissues?

Detecting OR9G1 in non-olfactory neural tissues presents several methodological challenges that researchers must address:

• Low expression levels: Olfactory receptors in non-olfactory tissues generally have lower expression levels compared to the olfactory epithelium, requiring highly sensitive detection methods .

• Cellular heterogeneity: Brain tissues contain diverse cell populations, and ORs may be expressed in specific neuronal subtypes, necessitating techniques to isolate or identify these specific cells .

• Co-localization complexity: Determining whether all components of the receptor signaling pathway are present in the same cell requires sophisticated co-localization studies .

• Multiple OR expression: Unlike olfactory neurons that typically express one OR type, neurons outside the olfactory epithelium may express multiple ORs, complicating the interpretation of expression data .

• Transcriptional regulation differences: The mechanism of OR gene transcriptional regulation may differ between olfactory epithelia and brain neurons, requiring different analytical approaches .

To overcome these challenges, researchers have successfully employed techniques such as laser capture microdissection coupled with nano cap-analysis of gene expression (nanoCAGE) technology to isolate specific neuronal populations, as demonstrated in studies of mesencephalic dopaminergic neurons in mice . Additionally, functional validation through calcium imaging in isolated primary neurons has proven effective for confirming OR activity in non-olfactory neurons .

How does the lyophilization process impact the performance of HRP-conjugated OR9G1 antibodies?

Lyophilization significantly enhances the performance of HRP-conjugated antibodies, including those targeting olfactory receptors like OR9G1. Research has demonstrated that introducing a lyophilization step after activating HRP (through sodium meta periodate oxidation of carbohydrate moieties) and before conjugation with antibodies substantially improves conjugate sensitivity and stability .

The lyophilization process impacts HRP-conjugated antibody performance through several mechanisms:

• Enhanced binding capacity: Lyophilization of activated HRP increases the number of HRP molecules that can bind to each antibody molecule, creating a poly-HRP nature that amplifies signal generation .

• Improved reaction kinetics: By reducing reaction volume without changing the amount of reactants, lyophilization increases the collision frequency between antibody molecules and activated HRP molecules according to collision theory, leading to more efficient conjugation .

• Extended shelf-life: Lyophilized activated HRP can be maintained at 4°C for longer periods, improving the practicality of conjugate preparation .

• Significantly increased sensitivity: Studies have shown that HRP-antibody conjugates prepared with lyophilized activated HRP can be used at dilutions of 1:5000, while conventional methods without lyophilization typically require much higher concentrations (dilutions as low as 1:25) .

• Preserved enzymatic activity: The modified method preserves the enzymatic activity of HRP while maximizing conjugation efficiency, as confirmed through UV spectrophotometry, SDS-PAGE analysis, and functional ELISA testing .

These improvements are particularly valuable for OR9G1 research, where detecting potentially low expression levels in non-olfactory tissues requires highly sensitive antibodies.

What are the optimal storage and handling conditions for maintaining OR9G1 Antibody, HRP conjugated activity?

To maintain optimal activity of OR9G1 Antibody, HRP conjugated, researchers should adhere to the following storage and handling recommendations:

• Storage temperature: Upon receipt, store the antibody at -20°C or -80°C for long-term stability .

• Freeze-thaw cycles: Avoid repeated freeze-thaw cycles as these can lead to denaturation and loss of activity. Aliquoting the antibody upon first thaw is strongly recommended .

• Working dilutions: Prepare working dilutions immediately before use and maintain them in appropriate buffer conditions (typically PBS with 50% glycerol) .

• Storage buffer composition: The antibody is supplied in a buffer containing 0.03% Proclin 300 as a preservative and 50% Glycerol in 0.01M PBS at pH 7.4. This composition should be considered when designing experiments to avoid buffer incompatibilities .

• Light exposure: Minimize exposure to light, particularly when working with the HRP-conjugated form, as HRP is light-sensitive.

• Contamination prevention: Use sterile techniques when handling the antibody to prevent microbial contamination.

• Transportation: If transportation between laboratories is necessary, maintain cold chain conditions using dry ice or similar methods.

Proper storage and handling are essential for maintaining the antibody's specificity and sensitivity, particularly for detecting potentially low levels of OR9G1 in non-olfactory tissues where signal optimization is crucial.

How can researchers validate the specificity of OR9G1 Antibody, HRP conjugated in experimental systems?

Validating antibody specificity is crucial for reliable OR9G1 research. Researchers should implement the following comprehensive validation strategy:

• Positive controls:

  • Use tissues/cells known to express OR9G1 (based on mRNA data), such as olfactory epithelium

  • Include recombinant OR9G1 protein at known concentrations

  • Consider heterologous cells transfected with OR9G1 expression constructs (similar to methods used for Olfr287 validation)

• Negative controls:

  • OR9G1 knockout models (if available)

  • Tissues known not to express OR9G1

  • Pre-adsorption control with immunizing peptide (amino acids 290-304 of human OR9G1)

  • Primary antibody omission controls

• Cross-reactivity assessment:

  • Test against closely related olfactory receptors, particularly other OR9G family members

  • Western blot analysis to confirm detection of protein at the expected molecular weight

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

• Correlation verification:

  • Compare protein detection patterns with mRNA expression (RT-PCR, in situ hybridization)

  • Use multiple antibodies targeting different epitopes of OR9G1 when available

  • Implement orthogonal detection methods such as RNA-Scope in parallel with immunodetection

• Functional validation:

  • Similar to experiments with Olfr287, consider calcium imaging in cells expressing OR9G1 upon stimulation with putative ligands when designing a complete validation strategy

Thorough validation is especially important for olfactory receptors like OR9G1 due to their expression in unexpected locations and potential functional diversity in the central nervous system.

What are the recommended ELISA protocols for optimal detection of OR9G1 using HRP-conjugated antibodies?

Based on research with HRP-conjugated antibodies and specific information about OR9G1 Antibody, HRP conjugated, the following optimized ELISA protocol is recommended:

Direct ELISA Protocol for OR9G1 Detection:

• Coating:

  • Coat high-binding 96-well plates with purified OR9G1 protein or tissue/cell lysates containing OR9G1 (typically 1-10 μg/ml) in carbonate-bicarbonate buffer (pH 9.6)

  • Incubate overnight at 4°C in a humid chamber

• Blocking:

  • Wash wells 3 times with PBS containing 0.05% Tween-20 (PBST)

  • Block with 2-5% BSA in PBS for 1-2 hours at room temperature

• Primary Antibody:

  • Apply OR9G1 Antibody, HRP conjugated directly to wells

  • Start with a dilution range of 1:1000 to 1:5000 based on enhanced sensitivity of properly conjugated HRP-antibodies

  • Incubate for 1-2 hours at room temperature or overnight at 4°C

• Detection:

  • Wash 4-5 times with PBST to remove unbound antibody

  • Add appropriate HRP substrate (TMB, ABTS, or enhanced chemiluminescent substrates)

  • For TMB, incubate for 15-30 minutes at room temperature protected from light

  • Stop the reaction with 2N H₂SO₄ if using colorimetric substrates

• Measurement:

  • For colorimetric detection, read absorbance at appropriate wavelength (450nm for TMB)

  • For chemiluminescence, read using a luminometer

• Controls to include:

  • Positive control: Recombinant OR9G1 protein

  • Negative control: Wells without primary antibody

  • Background control: Wells with irrelevant protein coating

• Optimization considerations:

  • If signal is weak, consider using enhanced chemiluminescent substrates for greater sensitivity

  • Further optimization may include extending incubation times or adjusting antibody concentrations

  • For detecting OR9G1 in brain tissue samples, additional sample preparation steps such as membrane protein enrichment may be necessary

This protocol leverages the advantages of HRP-conjugated antibodies, which have demonstrated significantly improved sensitivity (p value < 0.001) compared to classical conjugation methods .

How can researchers troubleshoot common issues when using OR9G1 Antibody, HRP conjugated in experimental applications?

The following troubleshooting guide addresses common issues encountered when using OR9G1 Antibody, HRP conjugated, particularly in ELISA and other applications:

Table 1: Troubleshooting Guide for OR9G1 Antibody, HRP Conjugated Applications

When troubleshooting specifically for OR9G1 detection in neural tissues, researchers should consider that olfactory receptors often show lower expression in non-olfactory tissues compared to the olfactory epithelium . This may require additional sensitivity optimization, including signal amplification strategies or more concentrated samples.

How can OR9G1 antibodies contribute to understanding olfactory receptor functions in neurodegenerative diseases?

OR9G1 antibodies are valuable tools for investigating the emerging roles of olfactory receptors in neurodegenerative diseases. Research has established that olfactory receptor gene expression is altered in several neurodegenerative conditions including Parkinson's disease, Alzheimer's disease, progressive supranuclear palsy, and sporadic Creutzfeldt-Jakob disease with disease-, region- and subtype-specific patterns .

The specific contributions of OR9G1 antibodies to this field include:

• Mapping expression patterns: OR9G1 antibodies enable detailed mapping of receptor expression in different brain regions and how these patterns change during disease progression. This is particularly important since olfactory dysfunction often precedes other symptoms in neurodegenerative diseases .

• Identifying cell-type specific expression: Using OR9G1 antibodies in co-localization studies with neuronal markers can reveal which specific neuronal populations express this receptor, providing insights into potential functional roles .

• Monitoring disease progression: Changes in OR9G1 protein levels could serve as potential biomarkers for disease progression, complementing studies that have already identified alterations in other OR family members in conditions like Alzheimer's disease .

• Investigating signaling pathways: OR9G1 antibodies can help elucidate whether the canonical olfactory signaling components (Gαolf, AC3) are co-expressed in the same cells, providing insights into potential signaling mechanisms in non-olfactory neurons .

• Developing therapeutic approaches: As olfactory receptors can function as chemoreceptors that react to endogenous ligands, understanding OR9G1 expression and function could potentially lead to the development of novel therapeutic approaches targeting these receptors .

Future studies should focus on correlating OR9G1 expression changes with clinical symptoms and investigating potential downstream effects of OR9G1 activation or inhibition in neural tissues affected by neurodegenerative processes.

What advanced techniques can be combined with OR9G1 antibody detection for comprehensive functional studies?

To conduct comprehensive functional studies of OR9G1, researchers can combine OR9G1 antibody detection with several advanced techniques:

• Single-cell transcriptomics with spatial resolution:

  • Combine single-cell RNA sequencing with in situ antibody detection to correlate OR9G1 protein expression with transcriptional profiles

  • Implement spatial transcriptomics techniques to map OR9G1 expression within specific brain regions while preserving tissue architecture

• CRISPR-Cas9 gene editing:

  • Generate OR9G1 knockout or knock-in reporter models to study loss-of-function or track expression

  • Create point mutations to study structure-function relationships

  • Use CRISPR-based epigenetic modifiers to study regulation of OR9G1 expression

• Live-cell imaging with functional readouts:

  • Employ calcium imaging in neurons expressing OR9G1 to detect activity upon stimulation with potential ligands, similar to methods used for Olfr287

  • Implement FRET-based sensors to monitor protein-protein interactions involving OR9G1

  • Utilize genetically encoded voltage indicators to correlate OR9G1 activation with neuronal activity

• Optogenetics and chemogenetics:

  • Combine OR9G1 detection with optogenetic manipulation of specific neuronal populations

  • Implement DREADD (Designer Receptors Exclusively Activated by Designer Drugs) technology to modulate signaling in OR9G1-expressing neurons

• High-throughput ligand screening:

  • Develop heterologous expression systems for OR9G1 combined with functional readouts

  • Screen libraries of endogenous metabolites and small molecules to identify potential ligands

  • Validate findings in primary neuronal cultures using OR9G1 antibodies to confirm expression

• Structural biology approaches:

  • Utilize cryo-electron microscopy to determine the three-dimensional structure of OR9G1

  • Apply molecular modeling to design specific agonists or antagonists

  • Implement biophysical techniques to study receptor-ligand interactions

• Translational approaches:

  • Analyze postmortem human brain samples from patients with neurodegenerative diseases using OR9G1 antibodies

  • Correlate findings with clinical data and disease progression

  • Develop potential biomarkers based on OR9G1 expression patterns

These combined approaches would provide a comprehensive understanding of OR9G1's functional roles in the nervous system and its potential involvement in neurological disorders.

What considerations should researchers make when planning longitudinal studies using OR9G1 antibodies in neurodegeneration models?

When planning longitudinal studies using OR9G1 antibodies in neurodegeneration models, researchers should carefully consider the following aspects:

• Experimental timeline design:

  • Include multiple time points beginning before symptom onset through disease progression

  • For transgenic models like APP/PS1 mice, start measurements from 3 months (early stage) through 12 months (advanced stage) based on known olfactory receptor expression changes

  • Include age-matched controls at each time point to account for age-related changes independent of pathology

• Antibody consistency and validation:

  • Use antibodies from the same lot throughout the study if possible

  • Validate antibody specificity at each time point, as tissue properties change with disease progression

  • Include appropriate positive and negative controls at each stage

• Complementary measurements:

  • Combine protein detection with mRNA analysis to distinguish between transcriptional and post-transcriptional changes

  • Include assessments of downstream signaling molecules like adenylyl cyclase 3 (AC3) and Gαolf

  • Correlate OR9G1 expression with established biomarkers of neurodegeneration

• Region-specific analyses:

  • Sample multiple brain regions at each time point, as expression changes may vary regionally

  • Include olfactory epithelium analysis for comparison with non-olfactory neural tissues

  • Focus on regions known to be affected early in the specific neurodegenerative disease model

• Functional correlations:

  • Include behavioral assessments of olfactory function at each time point

  • Correlate OR9G1 expression changes with cognitive or motor symptoms depending on the model

  • Consider in vivo functional imaging to track changes in neural activity

• Technical considerations:

  • Standardize tissue collection, processing, and storage procedures

  • Use consistent immunohistochemistry protocols and imaging parameters

  • Implement automated quantification methods to reduce bias

  • Consider tissue clearing techniques for whole-brain 3D imaging of OR9G1 expression

• Sample size and statistical planning:

  • Calculate appropriate sample sizes based on expected effect sizes

  • Plan for attrition, especially in longitudinal studies with aging animals

  • Use appropriate statistical methods for repeated measures and multiple comparisons

• Translational relevance:

  • Include analyses of human postmortem samples at different disease stages when available

  • Consider how findings in animal models relate to human disease progression

  • Evaluate potential for OR9G1 as a biomarker or therapeutic target

By carefully planning longitudinal studies with these considerations, researchers can generate robust data on how OR9G1 expression and function change throughout disease progression, potentially revealing new insights into the role of olfactory receptors in neurodegenerative processes.

What are the emerging research directions for OR9G1 and other olfactory receptors in non-chemosensory contexts?

Research on OR9G1 and other olfactory receptors in non-chemosensory contexts is evolving rapidly, with several promising directions emerging:

• Deciphering ligand diversity: Future research should focus on identifying endogenous ligands for OR9G1 and other ORs in the brain. Unlike in the olfactory epithelium, potential ligands in the brain could include molecules delivered through the bloodstream, cerebrospinal fluid, neighboring neurons and glial cells, or even intracellular metabolites . High-throughput screening approaches combined with structural biology will be essential for this discovery process.

• Elucidating neural network functions: Understanding how OR9G1 activation influences neuronal activity patterns and network dynamics represents a significant research frontier. This includes investigating whether OR9G1 modulates synaptic transmission, neuronal excitability, or circuit-level information processing.

• Therapeutic applications: The specific expression patterns of ORs in the brain, combined with their potential as targets for small molecule drugs, positions them as candidates for novel therapeutic approaches. Design and isolation of small molecules acting as cell-type specific agonists or antagonists for selected ORs represents a promising avenue .

• Biomarker development: Changes in OR expression patterns in neurodegenerative diseases suggest potential applications as biomarkers. Research should explore whether OR9G1 expression changes appear early in disease progression and whether they can be detected in accessible biofluids.

• 3D receptor structure determination: Reconstruction of the three-dimensional structure of receptors like OR9G1 will advance the analysis of activation mechanisms and facilitate the recognition and design of functional ligands, including specific agonists and antagonists . This represents a challenging but potentially transformative research direction.

• Investigating OR cross-talk: Future studies should explore how multiple ORs expressed in the same neuron (unlike in the olfactory epithelium) interact and potentially influence each other's signaling pathways .

• Developmental dynamics: Research into how OR expression patterns change during development and aging will provide insights into their fundamental roles in the central nervous system.

These emerging directions will expand our understanding of the non-canonical roles of olfactory receptors like OR9G1 in the nervous system and may reveal unexpected therapeutic opportunities for neurological disorders.

How might advances in antibody technology improve future OR9G1 research applications?

Advances in antibody technology are poised to significantly enhance OR9G1 research in several key ways:

• Recombinant antibody development: Moving beyond traditional polyclonal antibodies to recombinant monoclonal antibodies targeting specific OR9G1 epitopes will improve reproducibility and specificity. These antibodies can be engineered for optimal affinity and reduced cross-reactivity with other olfactory receptor family members.

• Nanobodies and single-domain antibodies: These smaller antibody fragments offer improved tissue penetration and access to conformational epitopes that might be inaccessible to conventional antibodies. Their small size makes them particularly valuable for studying membrane proteins like OR9G1 in intact tissues.

• Proximity labeling antibodies: Conjugating OR9G1 antibodies with enzymes like APEX2, HRP, or TurboID will enable identification of proteins in close proximity to OR9G1 in living cells, helping to map the receptor's interactome and signaling partners.

• Conditional antibody activation: Light-activatable or chemically-induced dimerization systems coupled to antibody fragments will allow temporal control of OR9G1 detection or manipulation in specific tissues or cells.

• Multiparametric detection systems: Development of multiplexed antibody panels combining OR9G1 detection with markers for specific cell types and signaling molecules will provide more comprehensive data from single experiments. Technologies like Imaging Mass Cytometry or CODEX can simultaneously visualize dozens of markers.

• Enhanced HRP conjugation methods: Building on research demonstrating the benefits of lyophilization in HRP-antibody conjugation, further refinements in conjugation chemistry will continue to improve sensitivity. Studies have shown that modified conjugation protocols can enhance sensitivity by orders of magnitude (from 1:25 to 1:5000 dilutions) .

• In vivo compatible antibody formats: Development of antibodies that can cross the blood-brain barrier or be expressed intracellularly will enable dynamic in vivo imaging of OR9G1 in animal models.

• Computationally designed antibodies: AI-assisted antibody design targeting specific OR9G1 epitopes will accelerate development of highly specific research tools.