OR9G1 Antibody, FITC conjugated

What is OR9G1 and what cellular functions does it mediate?

OR9G1 (Olfactory receptor 9G1) belongs to the family of G-protein-coupled receptors and functions primarily as an odorant receptor. Like other olfactory receptors discovered in non-sensory tissues, OR9G1 may mediate various cellular functions beyond olfaction. Similar olfactory receptors have been shown to influence cellular homeostasis through signaling pathways involving cytosolic Ca²⁺ and cAMP elevation. For example, the related olfactory receptor OR51E2 has been demonstrated to influence melanocyte proliferation, melanogenesis, and dendritogenesis when activated by appropriate ligands . While the specific cellular functions of OR9G1 outside the olfactory system remain under investigation, research frameworks established with other olfactory receptors provide valuable models for understanding potential cellular roles.

What are the basic characteristics of the OR9G1 Antibody, FITC conjugated?

The OR9G1 Antibody, FITC conjugated is a polyclonal antibody raised in rabbits against a recombinant Olfactory receptor 9G1 protein fragment (amino acids 290-304). It has the following specifications:

• Clonality: Polyclonal

• Host species: Rabbit

• Target: Human OR9G1 (UniProt ID: Q8NH87)

• Isotype: IgG

• Conjugate: FITC (Fluorescein isothiocyanate)

• Purity: >95%, Protein G purified

• Format: Liquid

• Buffer composition: 0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4

This antibody is particularly useful for fluorescence-based detection methods due to its FITC conjugation, enabling direct visualization without the need for secondary antibodies.

How should the OR9G1 Antibody, FITC conjugated be stored and handled?

For optimal preservation of antibody activity, the OR9G1 Antibody, FITC conjugated should be stored at -20°C or -80°C immediately upon receipt. Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of antibody function . When working with the antibody:

• Aliquot the antibody upon first thaw to minimize freeze-thaw cycles

• When removing from storage, thaw on ice or at 4°C

• Handle the antibody under subdued light conditions as FITC is photosensitive

• Store working dilutions at 4°C for short-term use (1-2 weeks)

• Centrifuge the vial briefly before opening to ensure collection of all material

Proper storage and handling are critical for maintaining antibody performance and experimental reproducibility.

How can I validate the specificity of OR9G1 Antibody, FITC conjugated in my experimental system?

Validating antibody specificity is crucial for reliable results. For OR9G1 Antibody, FITC conjugated, consider implementing these validation strategies:

• Positive and negative controls: Include tissues/cells known to express OR9G1 as positive controls and those lacking expression as negative controls. Based on the olfactory receptor expression patterns, olfactory epithelium would serve as a positive control.

• siRNA knockdown validation: Similar to methods used for validating other olfactory receptor antibodies, transfect cells with OR9G1-specific siRNA and scrambled control siRNA. Reduction in staining intensity with the target-specific siRNA but not the control would confirm antibody specificity .

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide (amino acids 290-304 of OR9G1) before application to samples. This should abolish specific staining.

• Western blot analysis: Verify single band detection at the expected molecular weight (approximately 35-40 kDa) in positive control samples, with band disappearance following siRNA knockdown.

• Heterologous expression systems: Similar to methods used for other olfactory receptors, express OR9G1 in Hana3a cells (which have been optimized for olfactory receptor expression) and confirm antibody detection .

What is the relationship between OR9G1 and other olfactory receptors like OR51E2 in terms of signaling pathways?

Olfactory receptors typically signal through G protein-coupled pathways, but their specific downstream effects can vary. While direct evidence for OR9G1 signaling is limited in the provided sources, research on related receptors like OR51E2 provides insight into potential signaling mechanisms:

• Calcium signaling: OR51E2 activation leads to elevated cytosolic Ca²⁺, which mediates cellular responses like melanogenesis. Similar mechanisms might exist for OR9G1 .

• cAMP pathways: Olfactory receptors commonly signal through cAMP elevation. OR51E2 activation demonstrates this pathway's involvement in cellular differentiation .

• MAPK signaling: While not directly confirmed for OR9G1, olfactory receptors may activate MAPK pathways, as evidenced by studies examining p44/42 MAPK and p38 MAPK phosphorylation in response to receptor activation .

Understanding these signaling parallels may help guide investigations into OR9G1-specific pathways, though direct experimental confirmation is necessary to establish OR9G1's signaling profile.

How can systems serology approaches be applied to study antibody interactions with OR9G1?

Systems serology offers powerful tools for comprehensively characterizing antibody properties and interactions. For studying OR9G1 antibodies, researchers could adapt approaches used for other immune studies:

• Fc-binding protein arrays: These can assess binding to various Fc receptors (FcGRIIA, FcGRIIB, FcGRIIIA, and FcGRIIIB) and complement components like C1q, revealing functional properties beyond simple antigen binding .

• Isotype and subclass profiling: Determine the distribution of IgG1-4, IgM, and IgA1-2 responses against OR9G1, which influences effector functions .

• Functional assays: Measure OR9G1-specific antibody-dependent cellular phagocytosis, NK cell activation, and complement deposition to characterize functional responses .

• Longitudinal analysis: Track antibody responses over time to understand the durability and evolution of responses, similar to approaches used in vaccine studies .

• Multivariate data integration: Apply computational approaches like LASSO-based feature selection and PLS-DA (Partial Least Squares Discriminant Analysis) to identify key antibody features that correlate with functional outcomes .

These systems approaches would provide a multidimensional view of OR9G1 antibody responses beyond what traditional single-parameter assays can reveal.

What are optimal protocols for using OR9G1 Antibody, FITC conjugated in immunofluorescence studies?

For optimal immunofluorescence results with OR9G1 Antibody, FITC conjugated, consider the following protocol guidelines:

• Sample preparation:

  • For cultured cells: Fix with 4% paraformaldehyde (10-15 minutes at room temperature)

  • For tissue sections: Use fresh-frozen sections or paraffin-embedded sections with appropriate antigen retrieval

• Blocking and permeabilization:

  • Block with 5-10% normal serum (from species unrelated to antibody host) in PBS with 0.1-0.3% Triton X-100 for 1 hour at room temperature

  • For membrane proteins like ORs, mild permeabilization may be preferable (0.1% Triton X-100 for 5-10 minutes)

• Antibody incubation:

  • Dilute antibody in blocking buffer (starting dilution 1:50-1:200, optimize as needed)

  • Incubate overnight at 4°C in humidified chamber

  • Protect from light due to FITC sensitivity

• Washing and counterstaining:

  • Wash 3x with PBS (5 minutes each)

  • Counterstain nuclei with DAPI (1 μg/mL, 5 minutes)

  • Mount with anti-fade mounting medium

• Controls and validation:

  • Include peptide competition controls

  • Perform parallel staining with alternate antibodies against the same target

  • Use appropriate positive control tissues

For co-localization studies, similar approaches to those used with OR51E2 can be applied, such as co-staining with markers like EEA-1 for early endosomes to determine subcellular localization .

How can I optimize calcium imaging experiments to study OR9G1 functionality?

Calcium imaging is valuable for studying the functionality of olfactory receptors like OR9G1. Based on methodologies used for related receptors, consider the following optimization steps:

• Cell preparation:

  • Culture cells expressing OR9G1 (native or transfected) on appropriate imaging substrates

  • For heterologous expression, use specialized cells like Hana3a that support functional expression of olfactory receptors

• Calcium indicator loading:

  • Load cells with a calcium-sensitive dye (Fura-2AM at 2-5 μM for 30-45 minutes at room temperature)

  • Wash thoroughly to remove extracellular dye

• Experimental setup:

  • Mount cells in a perfusion chamber with physiological buffer (typically Ringer's solution)

  • Maintain constant temperature (ideally 37°C) and stable perfusion rates

  • Use a system allowing rapid solution changes

• Stimulus application:

  • Apply potential ligands sequentially with sufficient wash periods between applications

  • Include positive controls (e.g., ionomycin) and vehicle controls

  • Consider dose-response relationships by applying increasing concentrations

• Data analysis:

  • Calculate F340/F380 ratios for Fura-2 or ΔF/F0 for single-wavelength indicators

  • Compare response amplitudes, kinetics, and recovery times

  • Perform statistical analysis across multiple independent experiments

• Validation approaches:

  • Use siRNA knockdown of OR9G1 to confirm specificity of responses

  • Apply receptor antagonists to block responses

  • Include positive control stimuli that activate endogenous calcium pathways

What methods can be used to study OR9G1 subcellular localization and trafficking?

Understanding OR9G1 subcellular localization provides insights into its function and regulation. Based on approaches used for other olfactory receptors, consider these methods:

• Co-immunocytochemistry:

  • Co-stain with markers for specific cellular compartments:

    • EEA-1 for early endosomes

    • LAMP1 for lysosomes

    • GM130 for Golgi apparatus

    • Calnexin for endoplasmic reticulum

  • This approach has successfully revealed OR51E2 localization in early endosomes

• Subcellular fractionation:

  • Prepare plasma membrane, cytosolic, and organelle fractions

  • Analyze OR9G1 distribution by Western blotting

  • Confirm fraction purity with compartment-specific markers

• Live cell imaging:

  • Create GFP- or other fluorescent protein-tagged OR9G1 constructs

  • Perform time-lapse imaging to track receptor trafficking in response to stimuli

  • Quantify surface expression versus internalization rates

• Surface biotinylation assays:

  • Label surface proteins with membrane-impermeable biotin reagents

  • Isolate biotinylated proteins with streptavidin pulldown

  • Detect OR9G1 by Western blotting to quantify surface expression

• Electron microscopy:

  • Use immunogold labeling with OR9G1 antibodies

  • Provides high-resolution images of receptor localization at the ultrastructural level

These approaches can reveal whether OR9G1 primarily localizes to the plasma membrane, internal compartments, or undergoes stimulus-dependent trafficking, similar to observations made with OR51E2 .

What are common issues encountered with OR9G1 Antibody, FITC conjugated and how can they be resolved?

Researchers may encounter several challenges when working with OR9G1 Antibody, FITC conjugated. Here are common issues and their solutions:

• Weak or no signal:

  • Increase antibody concentration

  • Optimize fixation protocols (over-fixation can mask epitopes)

  • Try different antigen retrieval methods for tissue sections

  • Ensure sample contains OR9G1-expressing cells

  • Check fluorescence microscope settings (FITC excitation ~495nm, emission ~519nm)

  • Store antibody properly to prevent degradation

• High background:

  • Increase blocking time or concentration

  • Use more stringent washing (increase number or duration of washes)

  • Dilute antibody further

  • Include 0.1% BSA in washing buffers

  • Filter all solutions to remove particulates

• Non-specific binding:

  • Pre-adsorb antibody with tissue/cell powder from non-expressing samples

  • Validate with peptide competition assays

  • Increase blocking serum concentration

  • Use detergent-free buffers for membrane proteins to avoid epitope disruption

• Photobleaching:

  • Minimize exposure to light during all steps

  • Use anti-fade mounting media

  • Capture images quickly or use anti-photobleaching systems

  • Consider using lower intensity excitation

• Inconsistent results:

  • Standardize all protocols including fixation time and temperature

  • Aliquot antibody to avoid freeze-thaw cycles

  • Process all experimental samples simultaneously

  • Include internal controls in each experiment

How can I quantitatively analyze OR9G1 expression levels across different experimental conditions?

Quantitative analysis of OR9G1 expression requires reliable methods for signal measurement and normalization. Consider these approaches:

• Immunofluorescence quantification:

  • Capture images using identical acquisition parameters

  • Measure mean fluorescence intensity within defined regions of interest

  • Subtract background fluorescence from signal-free areas

  • Normalize to cell number using nuclear counterstains

  • Use software like ImageJ/FIJI, CellProfiler, or specialized image analysis platforms

• Flow cytometry:

  • Prepare single-cell suspensions and stain with OR9G1 Antibody, FITC

  • Measure mean fluorescence intensity per cell

  • Include isotype controls for gating strategies

  • Analyze distributions rather than just mean values

  • Consider dual staining with cell type-specific markers

• Western blot quantification:

  • Use appropriate loading controls (e.g., GAPDH, β-actin)

  • Perform densitometric analysis of bands

  • Ensure detection is in the linear range

  • Normalize to total protein using stain-free gels or Ponceau S

  • Use housekeeping proteins that don't change under your experimental conditions

• qRT-PCR for transcript levels:

  • Design specific primers for OR9G1

  • Validate primer efficiency using standard curves

  • Use reference genes that maintain stable expression

  • Apply ΔΔCT method for relative quantification

  • Consider absolute quantification with standard curves for more precise measurements

What methods can be used to determine if detected OR9G1 is functional in experimental samples?

Determining the functionality of OR9G1 requires assays that link receptor presence to downstream signaling events. Based on approaches used for other olfactory receptors, consider these functional assays:

• Calcium mobilization assays:

  • Use calcium imaging with Fura-2AM or other indicators

  • Apply potential ligands and measure calcium responses

  • Confirm specificity with receptor antagonists or siRNA knockdown

  • Quantify response amplitude, duration, and kinetics

• cAMP measurements:

  • Use ELISA-based cAMP assays or FRET-based reporters

  • Compare basal versus stimulated cAMP levels

  • Include forskolin as a positive control

  • Block cAMP degradation with phosphodiesterase inhibitors

• Downstream signaling activation:

  • Assess phosphorylation of p44/42 MAPK and p38 MAPK by Western blotting

  • Monitor activation kinetics following potential ligand application

  • Include positive controls that activate these pathways through other receptors

• Receptor internalization:

  • Measure surface receptor levels before and after ligand exposure

  • Track co-localization with endosomal markers like EEA-1

  • Quantify the rate and extent of internalization

• Functional cellular responses:

  • Identify cell type-specific responses (e.g., proliferation, differentiation)

  • Compare responses in OR9G1-expressing versus knockdown cells

  • Measure changes in gene expression profiles using qPCR arrays or RNA-seq

How can OR9G1 Antibody, FITC conjugated be used in neuroscience research?

OR9G1 Antibody, FITC conjugated offers multiple applications in neuroscience research:

• Olfactory system mapping:

  • Characterize OR9G1-expressing neurons in olfactory epithelium

  • Trace axonal projections to olfactory bulb glomeruli

  • Study zonal organization of olfactory receptor expression

  • Examine developmental regulation of OR9G1 expression

• Ectopic OR expression studies:

  • Detect OR9G1 in non-sensory tissues, similar to studies of OR51E2 in melanocytes

  • Investigate potential roles in other neural and non-neural tissues

  • Correlate expression with functional outcomes in various cell types

• Neural circuit analysis:

  • Identify potential OR9G1-responsive neurons in higher brain centers

  • Combine with functional imaging to map information flow

  • Use in circuit tracing when combined with retrograde labeling techniques

• Neurodevelopmental research:

  • Track OR9G1 expression during embryonic and postnatal development

  • Correlate with critical periods in sensory system formation

  • Study regeneration processes in olfactory neuroepithelium

• Comparative neurobiology:

  • Compare OR9G1 expression patterns across species

  • Explore evolutionary conservation of expression and function

  • Link to behavioral responses to specific odorants

What are the emerging applications of systems biology approaches to studying olfactory receptors like OR9G1?

Systems biology approaches are transforming our understanding of olfactory receptors, with several promising directions:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Create comprehensive models of OR signaling networks

  • Identify key regulatory nodes in olfactory processing

  • Similar to approaches used in systems serology for characterizing immune responses

• Single-cell analysis:

  • Apply single-cell RNA-seq to map OR expression at unprecedented resolution

  • Identify previously unknown cell types expressing OR9G1

  • Link receptor expression to cell-specific transcriptional programs

  • Track clonal relationships among OR-expressing cells

• Network modeling:

  • Develop mathematical models of OR signaling pathways

  • Predict responses to novel ligands

  • Identify cross-talk between different ORs

  • Apply LASSO-based feature selection to identify minimal elements required for specific functions

• Machine learning applications:

  • Predict ligand-receptor interactions based on receptor sequence

  • Classify ORs into functional groups

  • Develop automated image analysis for receptor localization studies

  • Integrate multiparametric data to identify key functional differences between receptor types

• Interactome mapping:

  • Identify OR9G1 protein-protein interactions

  • Map connections to various signaling pathways

  • Discover novel regulatory partners

  • Compare interaction networks across different olfactory receptors

What are the key considerations when designing experiments with OR9G1 Antibody, FITC conjugated?

When designing experiments with OR9G1 Antibody, FITC conjugated, researchers should consider several critical factors:

• Antibody validation: Thoroughly validate the antibody's specificity using multiple approaches including positive/negative controls, peptide competition, and siRNA knockdown to ensure reliable results .

• Experimental controls: Include appropriate controls for each experiment, such as isotype controls, secondary-only controls for background estimation, and positive controls from tissues known to express OR9G1.

• Sample preparation optimization: Different fixation and permeabilization methods can significantly affect antibody performance. Optimize these conditions specifically for OR9G1 detection in your experimental system.

• Quantification strategies: Plan robust quantification approaches in advance, ensuring appropriate statistical power and reproducibility of measurements across experimental replicates.

• Functional correlation: Where possible, link antibody-based detection to functional assays that confirm the biological activity of the detected receptor, similar to approaches used for other olfactory receptors .

• Technical limitations awareness: Recognize the inherent limitations of the antibody, including potential cross-reactivity with highly homologous olfactory receptors, sensitivity thresholds, and the qualitative nature of many immunostaining results.