OR9G1 Antibody, Biotin conjugated

Basic Research Questions

• What is OR9G1 and what is its biological role in olfactory perception?

  OR9G1 (olfactory receptor family 9 subfamily G member 1) is a member of the large family of G-protein-coupled receptors (GPCRs) encoded by a single coding-exon gene. Located on chromosome 11, OR9G1 (UniProt ID: Q8NH87) is involved in the recognition and G protein-mediated transduction of odorant signals in the nose . Like other olfactory receptors, OR9G1 contains a characteristic 7-transmembrane domain structure, similar to many neurotransmitter and hormone receptors . These receptors interact with specific odorant molecules to initiate a neuronal response that triggers the perception of smell, with each receptor having varying sensitivities to different odorants, contributing to the complex system of olfactory discrimination .

• How is OR9G1 Antibody, Biotin conjugated prepared, and what is its molecular structure?

  OR9G1 Antibody, Biotin conjugated is typically produced using recombinant Human Olfactory receptor 9G1 protein fragments (specifically the 290-304aa region) as the immunogen . The antibody production process involves immunizing rabbits with this specific epitope, followed by purification using Protein G chromatography to obtain >95% purity .

  The biotin conjugation process attaches biotin molecules to the antibody structure. In some preparations, a spacer is positioned between biotin and the antibody (sometimes referred to as Biotin-SP), which extends the biotin moiety away from the antibody surface to increase accessibility to binding sites on streptavidin . The final product is supplied in liquid form in a buffer containing preservative (0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) .

• What are the optimal storage conditions for maintaining OR9G1 Antibody, Biotin conjugated activity?

  For maintaining optimal activity of OR9G1 Antibody, Biotin conjugated, the recommended storage conditions are:

  • Store at -20°C or -80°C upon receipt

  • Avoid repeated freeze/thaw cycles as this can denature the antibody and reduce its efficacy

  • For longer-term storage, aliquot the antibody into smaller volumes before freezing to minimize freeze/thaw cycles

  • The antibody is typically formulated in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative, which helps maintain stability during storage

  Proper storage is critical for maintaining the binding activity of the antibody, as improper handling can lead to denaturation and loss of specificity in experimental applications.

• What experimental applications is OR9G1 Antibody, Biotin conjugated validated for?

  OR9G1 Antibody, Biotin conjugated has been primarily validated for Enzyme-Linked Immunosorbent Assay (ELISA) . While primarily tested for ELISA, related OR9G1 antibodies with different conjugates have been reported to be suitable for additional applications including:

  • Western Blotting (WB)

  • Immunofluorescence (IF)

  • Immunocytochemistry (ICC)

  Each application requires specific optimization parameters, and researchers should validate the antibody for their specific experimental conditions. As with all research antibodies, preliminary testing and optimization are recommended when adapting the antibody to applications beyond those explicitly validated by the manufacturer .

Advanced Research Questions

• What are the key experimental considerations when validating OR9G1 Antibody, Biotin conjugated specificity?

  Validating antibody specificity is critical for ensuring reliable experimental results. For OR9G1 Antibody, Biotin conjugated, consider these methodological approaches:

  1. Positive and negative controls:

     • Use known OR9G1-expressing tissues/cells as positive controls

     • Include tissues/cells known not to express OR9G1 as negative controls

     • Consider using OR9G1 knockout or knockdown models if available

  2. Cross-reactivity assessment:

     • Test against related olfactory receptors, particularly those with high sequence homology

     • Given that humans have approximately 400 intact odorant receptors with sequence similarities , cross-reactivity testing is particularly important

  3. Peptide competition assays:

     • Pre-incubate the antibody with excess immunogenic peptide (290-304aa of OR9G1)

     • The specific signal should be significantly reduced or eliminated

  4. Multiple detection methods:

     • Compare results using different detection techniques (e.g., ELISA, Western blot, immunofluorescence)

     • Consistent results across multiple techniques strengthen validation

  5. Antibody titration:

     • Perform dose-response experiments to determine optimal concentration

     • A specific antibody should show a clear titration curve with plateau

  The specificity of antibodies against olfactory receptors is particularly challenging due to the high sequence homology within this large gene family, making thorough validation essential .

• How can experimental design mitigate signal-to-noise challenges when using OR9G1 Antibody, Biotin conjugated in neuronal tissue studies?

  Working with neuronal tissues presents unique challenges for olfactory receptor detection. To optimize signal-to-noise ratio when using OR9G1 Antibody, Biotin conjugated:

  1. Tissue preparation optimization:

     • Use fresh or properly fixed tissues (optimize fixation time and conditions)

     • For olfactory epithelium, consider specialized fixation protocols that preserve epitope integrity while maintaining tissue architecture

  2. Blocking optimization:

     • Implement stringent blocking protocols using 5-10% normal serum from the same species as the secondary detection reagent

     • Consider adding 0.1-0.3% Triton X-100 for improved permeabilization in immunohistochemistry applications

  3. Signal amplification strategies:

     • Leverage the biotin-streptavidin system for signal amplification

     • Using streptavidin conjugated to enzymes like alkaline phosphatase can significantly enhance detection sensitivity

     • For fluorescence applications, consider using streptavidin conjugated to bright, photostable fluorophores

  4. Reduce autofluorescence:

     • Pre-treat sections with sodium borohydride or commercial autofluorescence quenchers

     • Use longer wavelength fluorophores to avoid tissue autofluorescence (if using fluorescence detection)

  5. Optimize streptavidin detection:

     • When using Biotin-SP-conjugated antibodies with alkaline phosphatase-conjugated streptavidin, the 6-atom spacer extends the biotin away from the antibody surface, making it more accessible to binding sites on streptavidin

  6. Control for endogenous biotin:

     • Block endogenous biotin in tissues using avidin/biotin blocking kits before applying the primary antibody

  These methodological refinements are particularly important when studying low-abundance GPCRs like OR9G1 in complex neuronal tissues .

• How does experimental troubleshooting differ between various biotin-conjugated antibody detection systems when using OR9G1 Antibody?

  Different detection systems for biotin-conjugated OR9G1 antibody present unique troubleshooting challenges:

  Detection System	Common Issues	Troubleshooting Approach
  Streptavidin-HRP	High background	1. Increase blocking stringency with biotin-free BSA 2. Add 0.05-0.1% Tween-20 to wash buffers 3. Verify HRP activity with control substrates
  Streptavidin-Alkaline Phosphatase	Low signal sensitivity	1. Extend substrate development time 2. Use Biotin-SP conjugate for improved binding 3. Optimize buffer pH (optimal range: 8.0-9.5)
  Streptavidin-Fluorophore	Photobleaching	1. Minimize exposure to light 2. Use anti-fade mounting media 3. Consider using quantum dot conjugates for greater photostability
  Streptavidin-Gold	Poor resolution	1. Optimize gold particle size 2. Use silver enhancement for improved visualization 3. Verify protein concentration and gold conjugation efficiency

  System-specific verification steps include:

  1. Check antibody concentration: Verify using Bradford protein assay or NanoDrop Spectrophotometer

  2. Assess biotin conjugation efficiency: Poor conjugation yield may result from incorrect initial antibody concentration or presence of interfering preservatives like Proclin

  3. Consider protein carriers: Remove protein carriers like BSA or gelatin that may interfere with biotin modification using affinity chromatography

  For challenging applications with low signal, consider implementing sequential detection systems where streptavidin-biotin interactions are used to build detection complexes with enhanced sensitivity .

• What are the methodological considerations for using OR9G1 Antibody, Biotin conjugated in conjunction with genetic variation studies of olfactory receptors?

  Combining antibody-based detection with genetic variation analysis requires careful experimental design:

  1. Correlation with genetic polymorphisms:

     • Human odorant receptor genes exhibit high genetic variability with significant functional consequences

     • Approximately 63% of odorant receptors have polymorphisms that alter function, with 11% being hyperfunctional, 6.8% hypofunctional, and 7.9% pseudogenes

     • Design experiments to correlate OR9G1 protein expression levels (detected via the biotin-conjugated antibody) with specific genetic variants

  2. Epitope variation considerations:

     • The OR9G1 antibody targets the 290-304aa region

     • Verify whether known genetic polymorphisms affect this epitope region through sequence analysis

     • Polymorphisms in or near the epitope may affect antibody binding affinity and generate false negatives

  3. Quantitative correlation methodology:

     • Implement parallel analysis of genetic variants (through sequencing) and protein expression (through antibody detection)

     • For human studies, consider using patient-derived samples with known OR9G1 genetic profiles

     • Normalize antibody signals to account for sample-to-sample variation

  4. Multiplex analysis approach:

     • Design experiments that simultaneously detect multiple olfactory receptors

     • Use OR9G1 Antibody, Biotin conjugated alongside antibodies for other olfactory receptors with different conjugates

     • Analyze co-expression patterns in relation to genetic variations

  5. Functional correlation:

     • Link antibody detection data with functional assays (e.g., calcium imaging, electrophysiology)

     • This approach can help determine whether genetic variations affecting protein levels also impact receptor function

  These methodologies are particularly valuable for understanding how the extensive genetic variability in human olfactory receptors translates to functional differences in olfactory perception .

• How can computational approaches be integrated with OR9G1 Antibody, Biotin conjugated experimental data to advance olfactory receptor research?

  Integration of experimental antibody data with computational methods can significantly enhance olfactory receptor research:

  1. Homology modeling and antibody epitope mapping:

     • Generate homology models of OR9G1 based on known GPCR structures using approaches similar to those used for OR1A1 and OR2W1

     • Use X-ray crystal structures of bovine rhodopsin (PDB ID: 1U19) as templates for homology modeling

     • Map the antibody's target epitope (290-304aa) onto the 3D model to visualize its structural context

     • This can help predict antibody accessibility in different experimental conditions

  2. Machine learning for agonist prediction:

     • Combine antibody-based receptor localization data with ML approaches similar to those used for OR1A1 and OR2W1

     • Random Forest (RF), Support Vector Machine (SVM), and Naïve Bayes (NB) classifiers have been successfully applied to predict olfactory receptor agonists

     • Train models using experimental activation data correlated with antibody-based expression levels

  3. Integrative data analysis:

     • Connect antibody-detected OR9G1 expression patterns with:
       a) Genomic data (SNPs, haplotypes)
       b) Transcriptomic data (RNA-seq expression levels)
       c) Functional data (calcium imaging responses to odorants)

     • Apply dimensionality reduction techniques to identify patterns across diverse datasets

  4. Molecular docking and virtual screening:

     • Use antibody validation data to confirm OR9G1 structural features for refined molecular docking

     • Apply virtual screening methods to identify potential novel ligands, similar to approaches used for OR51E2

     • Validate computational predictions through functional assays in OR9G1-expressing cells identified by antibody staining

  This integrated approach leverages both wet-lab antibody data and computational methods to accelerate discovery in the challenging field of olfactory receptor research .

• What methodological approaches can optimize the use of OR9G1 Antibody, Biotin conjugated in multiplexed detection systems?

  Multiplexed detection systems allow simultaneous analysis of multiple targets. For optimizing OR9G1 Antibody, Biotin conjugated in such systems:

  1. Orthogonal labeling strategies:

     • Pair biotin-conjugated OR9G1 antibody with antibodies against other targets using different conjugates (fluorophores, enzymes)

     • Ensure spectral separation or substrate differentiation to prevent signal overlap

     • Carefully control for potential cross-reactivity between detection systems

  2. Sequential detection protocol:

     • For co-localization studies, implement sequential staining protocols:
       a) Detect OR9G1 using the biotin-conjugated antibody and streptavidin-fluorophore
       b) Block remaining biotin binding sites with excess free biotin
       c) Proceed with additional antibody staining using different detection systems

  3. Tyramide signal amplification (TSA) integration:

     • Combine biotin-streptavidin with TSA for significantly enhanced sensitivity

     • This approach allows detection of low-abundance OR9G1 receptors

     • Critical for distinguishing genuine signal from background in complex tissue samples

  4. Advanced imaging considerations:

     • For spectral imaging, carefully characterize the emission/excitation profiles of all fluorophores

     • Implement spectral unmixing algorithms to separate overlapping signals

     • Use appropriate controls to establish detection thresholds

  5. Cross-validation strategy:

     • Perform parallel single-target detection alongside multiplexed detection

     • Compare signal intensities and distributions to validate multiplexed results

     • Address any discrepancies by optimizing antibody concentrations and detection parameters

  6. Custom detection optimization:

     • Develop custom combinations of biotin-binding proteins (streptavidin, avidin variants)

     • Adjust incubation times and temperatures for optimal signal development

     • Systematic testing of buffer compositions can significantly impact multistaining quality

  These methodological approaches enable robust multiplexed detection systems incorporating OR9G1 Antibody, Biotin conjugated, particularly valuable for studying olfactory receptor distribution patterns and co-expression with other neuronal markers .

• What are the critical considerations for designing experiments using OR9G1 Antibody, Biotin conjugated to study olfactory receptor trafficking?

  Studying olfactory receptor trafficking requires specialized experimental approaches:

  1. Subcellular localization analysis:

     • Implement high-resolution confocal or super-resolution microscopy to visualize OR9G1 trafficking

     • Co-stain with markers for specific cellular compartments (ER, Golgi, plasma membrane)

     • Use pulse-chase approaches with biotin-conjugated antibodies to track receptor movement

  2. Specialized fixation and permeabilization:

     • Optimize fixation protocols to preserve membrane structures (4% PFA with controlled fixation time)

     • Test different permeabilization agents (Triton X-100, saponin, digitonin) at varying concentrations

     • Saponin (0.01-0.05%) offers gentler permeabilization that better preserves membrane proteins

  3. Live-cell imaging adaptations:

     • For trafficking studies in live cells, consider using primary antibody fragments conjugated to cell-permeable biotin derivatives

     • Implement microfluidic systems for controlled antibody delivery during imaging

     • Combine with genetically-tagged OR9G1 constructs for validation

  4. Endocytic pathway analysis:

     • Design pulse-chase experiments to track internalization and recycling

     • Co-localize with endosomal markers (Rab5, Rab7, Rab11) to determine trafficking routes

     • Implement temperature blocks (4°C, 16°C, 20°C) to arrest trafficking at specific steps

  5. Trafficking in disease models:

     • Compare OR9G1 trafficking in normal vs. disease models

     • Recent findings in membrane trafficking components for antibody secretion provide methodological frameworks that can be adapted

  This experimental framework enables detailed investigation of how OR9G1 moves through the secretory and endocytic pathways, providing insights into the molecular mechanisms of olfactory receptor function .