OR10J3 Antibody

What is OR10J3 and why is it significant in olfactory research?

OR10J3 (olfactory receptor 10J3) is a 329 amino acid G protein-coupled receptor that localizes to the cilia of olfactory sensory neurons. The significance of this protein lies in its role within the olfactory system, where it binds to specific odor molecules, triggering a signal transduction cascade that propagates nerve impulses throughout the body . This process ultimately leads to transmission of signals to the brain and the perception of smell. OR10J3 is particularly noteworthy as it belongs to the largest gene family in the human genome - the olfactory receptor genes . The gene encoding OR10J3 maps to human chromosome 1, making it an important subject for both genomic and proteomic studies focused on sensory perception mechanisms .

What are the key differences between various OR10J3 antibody conjugates?

OR10J3 antibodies are available with several different conjugations, each optimized for specific experimental applications:

The choice between these conjugates depends primarily on your experimental setup, detection method, and potential co-localization studies . For multicolor immunofluorescence experiments, Alexa Fluor® conjugates typically offer superior photostability and brightness compared to traditional fluorophores, while unconjugated antibodies provide greater flexibility in detection methods when used with secondary antibodies .

How can I verify the specificity of OR10J3 antibodies in my experimental system?

Verifying antibody specificity is a critical step in experimental design. For OR10J3 antibodies, employ the following multi-tiered approach:

• Western blot validation: Run parallel lanes with positive control lysates (HUVEC or MCF-7 cells have been demonstrated to express OR10J3) . The expected molecular weight is approximately 36 kDa (calculated), though it may appear at ~72 kDa in some systems due to post-translational modifications or dimerization .

• Peptide blocking: Pre-incubate the antibody with the immunizing peptide (typically derived from C-terminal of human OR10J3) before application to your samples. This should abolish specific staining .

• Genetic validation: If possible, use OR10J3 knockout/knockdown models as negative controls.

• Cross-reactivity assessment: Test the antibody against tissues/cells from multiple species if cross-species reactivity is claimed by the manufacturer (common reactivity includes human and rat) .

• Immunofluorescence pattern analysis: OR10J3 should show characteristic membrane-associated staining patterns consistent with its role as a G protein-coupled receptor .

For antibodies claiming cross-reactivity with multiple species, validation in each species is essential, as epitope conservation may vary .

What are the optimal protocols for using OR10J3 antibodies in Western blot applications?

For optimal Western blot results with OR10J3 antibodies, follow this evidence-based protocol:

• Sample preparation:

  • Extract proteins from tissues/cells using RIPA buffer supplemented with protease inhibitors

  • Determine protein concentration (Bradford or BCA assay)

  • Load 20-50 μg total protein per lane

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels

  • Transfer to PVDF membrane (recommended over nitrocellulose for this target)

  • Confirm transfer efficiency with Ponceau S staining

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Incubate with primary OR10J3 antibody at 1:500-1:2000 dilution in 5% BSA/TBST overnight at 4°C

  • Wash 3× with TBST, 10 minutes each

  • Incubate with appropriate HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

• Detection and analysis:

  • Develop using ECL substrate

  • Expected molecular weight: 36 kDa (theoretical); may appear at ~72 kDa in some systems

  • Include positive control: HUVEC cell lysate has been validated

• Validation controls:

  • Peptide competition assay using the immunizing peptide

  • MCF-7 cell lysate can serve as additional positive control

This protocol has been optimized based on multiple peer-reviewed applications of OR10J3 antibodies in Western blotting .

How should OR10J3 antibodies be used for immunofluorescence applications?

For immunofluorescence applications using OR10J3 antibodies, follow this optimized protocol:

• Sample preparation:

  • For cultured cells: Grow cells on coverslips, fix with 4% paraformaldehyde for 15 minutes at room temperature

  • For tissue sections: Use freshly frozen or paraffin-embedded sections (4-6 μm thickness)

• Permeabilization and blocking:

  • Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

  • Block with 5-10% normal serum (from the same species as the secondary antibody) in PBS for 1 hour

• Antibody incubation:

  • For directly conjugated OR10J3 antibodies (Cy3, Alexa Fluor 488 or 555):

    • Dilute 1:50-1:200 in blocking buffer

    • Incubate overnight at 4°C in a humidified chamber

  • For unconjugated primary antibodies:

    • Dilute 1:200-1:1000 in blocking buffer

    • Incubate overnight at 4°C

    • Wash 3× with PBS

    • Incubate with appropriate fluorophore-conjugated secondary antibody

• Nuclear counterstaining and mounting:

  • Counterstain with DAPI (1 μg/ml) for 5 minutes

  • Mount using anti-fade mounting medium

• Imaging considerations:

  • OR10J3 should show membrane localization consistent with G protein-coupled receptors

  • For multi-color imaging with directly conjugated antibodies, ensure proper filter sets to avoid bleed-through

This protocol incorporates best practices from multiple sources and has been shown to produce specific staining in validated cell types .

What are the considerations for using OR10J3 antibodies in ELISA applications?

When employing OR10J3 antibodies in ELISA applications, consider the following critical parameters:

• Antibody dilution optimization:

  • Starting recommended dilution: 1:10000-1:20000

  • Perform checkerboard titration to determine optimal antibody concentration

  • Different lots may require slight adjustment of dilution factors

• Sample preparation considerations:

  • For cell/tissue lysates: Use gentle lysis buffers (avoid SDS or other strong detergents)

  • For serum samples: Pre-clear samples to remove potential interfering proteins

  • Consider spike-recovery experiments to validate sample matrix effects

• Assay validation parameters:

  • Determine lower limit of detection (LLOD) using serial dilutions

  • Establish assay linearity range

  • Cross-reactivity testing with related olfactory receptors

  • Inter- and intra-assay coefficient of variation should be <15%

• Controls and standards:

  • Include recombinant OR10J3 protein as standard curve

  • Run negative controls (null lysates) and blocking peptide controls

  • Consider adding recovery controls to validate sample processing

• Data interpretation considerations:

  • OR10J3 expression levels can vary significantly between tissue types

  • Potential for non-linear relationship between signal and protein concentration at very high or low concentrations

This comprehensive approach ensures robust and reproducible ELISA results when working with OR10J3 antibodies, based on standard biochemical practices and specific recommendations for this antibody class .

How can I address high background issues when using OR10J3 antibodies in immunostaining?

High background is a common challenge in immunostaining. When using OR10J3 antibodies, implement these evidence-based solutions:

• Optimize antibody dilution:

  • For direct conjugates (Cy3, Alexa Fluor), testing broader dilution ranges (1:25 to 1:400) may be necessary

  • For unconjugated antibodies in IF applications, try 1:400-1:1000 dilutions

  • Running parallel dilution series can quickly identify optimal concentration

• Modify blocking conditions:

  • Increase blocking time to 2 hours

  • Try alternative blocking agents: 2-5% BSA, commercial blocking solutions, or 0.1-0.3% fish gelatin

  • Add 5-10% serum from the same species as the secondary antibody to reduce non-specific binding

• Improve washing steps:

  • Increase wash buffer volume and duration (4-5 washes, 10 minutes each)

  • Add 0.1-0.3% Triton X-100 or 0.05-0.1% Tween-20 to wash buffers

  • Use gentle agitation during washing

• Control for autofluorescence:

  • Pre-treat samples with sodium borohydride (10 mg/ml for 15 minutes) to reduce autofluorescence

  • For tissues with high lipofuscin content, treatment with Sudan Black B (0.1-0.3% in 70% ethanol) can reduce autofluorescence

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies

  • Include a control with secondary antibody only

• Tissue-specific considerations:

  • For olfactory tissues: Additional peroxidase quenching may be necessary (3% H₂O₂ in methanol for 10 minutes)

  • For nasal epithelium: Extended blocking (3-4 hours) may improve specificity

These approaches address specific challenges encountered with OR10J3 antibodies and are based on empirical evidence from immunostaining applications .

Why might I observe discrepancies between the predicted and observed molecular weight of OR10J3 in Western blots?

The discrepancy between the predicted molecular weight of OR10J3 (approximately 36 kDa) and observed weight (sometimes around 72 kDa) is a commonly reported phenomenon that can be explained by several biological and technical factors:

• Post-translational modifications:

  • G protein-coupled receptors like OR10J3 often undergo extensive glycosylation, which can increase apparent molecular weight by 10-40 kDa

  • Phosphorylation sites on OR10J3 may alter migration patterns

  • Ubiquitination or SUMOylation can significantly affect molecular weight

• Protein dimerization and complexes:

  • OR10J3, like many GPCRs, can form stable homodimers resistant to SDS denaturation

  • Incomplete sample denaturation often allows dimers to persist on gels

  • Solution: Increase denaturation temperature to 95-98°C and extend boiling time to 10 minutes

• Technical considerations:

  • Gel percentage affects migration patterns (8% gels may better resolve higher molecular weight forms)

  • Buffer systems impact migration (Tris-glycine vs. Tris-tricine)

  • Protein markers should be validated with alternative marker systems

• Sample preparation factors:

  • Protease inhibition is critical during extraction to prevent artifactual bands

  • Different extraction buffers may solubilize different protein populations

• Validation approaches:

  • Run parallel lanes with different reduction conditions

  • Use epitope-tagged recombinant OR10J3 as size control

  • Consider 2D gel electrophoresis to resolve modifications

The observed 72 kDa band in some studies likely represents dimerized OR10J3 or heavily glycosylated forms that resist complete denaturation . Both the 36 kDa and 72 kDa bands may be specific depending on sample preparation methods.

What strategies can improve detection sensitivity when working with low-abundance OR10J3 expression?

Detecting low-abundance OR10J3 expression requires optimized strategies across sample preparation, antibody selection, and signal amplification:

• Sample enrichment techniques:

  • Immunoprecipitation before Western blotting can concentrate OR10J3 protein

  • Subcellular fractionation to isolate membrane fractions where OR10J3 localizes

  • For tissue samples, laser capture microdissection of olfactory epithelia increases specific content

• Antibody selection and handling:

  • Use high-affinity antibodies; compare different clones if available

  • Conjugated antibodies (Cy3, Alexa Fluor 488/555) typically offer 2-5× better detection threshold in IF applications

  • Extended primary antibody incubation (48-72 hours at 4°C) can improve weak signals

• Signal amplification methods:

  • For Western blots: ECL Prime or SuperSignal West Femto substrates increase sensitivity 10-50×

  • For IF/IHC: Tyramide signal amplification (TSA) systems can boost signal 10-200×

  • Biotin-streptavidin systems amplify signals approximately 4-8×

• Detection system optimization:

  • Cooled CCD cameras for fluorescence applications

  • Extended exposure times with low-noise imaging systems

  • Digital image accumulation (frame averaging) reduces noise

• Protocol modifications:

  • Reduce washing stringency slightly (use TBS instead of TBST for final washes)

  • Use polyvinyl alcohol-containing mounting media to reduce photobleaching

  • Consider tissue-specific antigen retrieval methods for improved epitope accessibility

• Quantification strategies:

  • Use digital image analysis with appropriate controls

  • Consider matched-exposure comparisons rather than variable exposures

  • Include concentration standards when possible

These approaches have collectively demonstrated improved detection of low-abundance G protein-coupled receptors, including olfactory receptors like OR10J3 .

How can OR10J3 antibodies be utilized in studies of olfactory receptor trafficking and localization?

Investigating OR10J3 trafficking and localization requires specialized approaches that leverage the specificity of antibodies:

• Pulse-chase immunofluorescence protocols:

  • Surface labeling with membrane-impermeable biotinylation reagents followed by internalization

  • Differential staining of surface vs. internalized receptors using non-permeabilized vs. permeabilized conditions

  • Recommended antibody concentration: 1:50-1:100 for Alexa Fluor conjugates

• Co-localization with trafficking markers:

  • Early endosome markers (EEA1, Rab5) for internalization

  • Recycling endosome markers (Rab11) for recycling pathways

  • ER/Golgi markers (calnexin, GM130) for biosynthetic pathways

  • Use dual-labeling with Alexa Fluor 488-conjugated OR10J3 antibodies and contrasting fluorophores for markers

• Live-cell imaging approaches:

  • Antibody fragments (Fab) conjugated to pH-sensitive fluorophores

  • Single-particle tracking of quantum dot-conjugated antibodies

  • FRAP (Fluorescence Recovery After Photobleaching) with labeled antibodies

• Super-resolution microscopy applications:

  • STORM/PALM imaging using directly conjugated antibodies

  • Structured illumination microscopy (SIM) for 2× resolution improvement

  • Optimal antibody dilution for super-resolution: 1:50 for conjugated antibodies

• Electron microscopy localization:

  • Immunogold labeling on freeze-substituted tissue

  • Cryo-immunoelectron microscopy

  • CLEM (Correlative Light and Electron Microscopy) for multi-scale context

These techniques can map the dynamic transport of OR10J3 from biosynthesis to functional localization in cilia and provide insights into the relationship between localization and function in olfactory sensory neurons .

What approaches can be used to study OR10J3 interaction partners using antibody-based techniques?

Studying the interactome of OR10J3 requires specialized antibody-based approaches to preserve physiologically relevant interactions:

• Co-immunoprecipitation strategies:

  • Native co-IP using membrane-solubilizing detergents (1% digitonin or 0.5% DDM)

  • Crosslinking-assisted co-IP to capture transient interactions (DSP, formaldehyde)

  • Recommended antibody amounts: 2-5 μg antibody per 500 μg protein lysate

  • HUVEC lysates serve as validated source material

• Proximity labeling approaches:

  • BioID or TurboID fusion proteins with OR10J3

  • APEX2 proximity labeling

  • Verification of interactions using reciprocal co-IP with OR10J3 antibodies at 1:500 dilution

• Protein complementation assays:

  • Split-GFP or split-luciferase with OR10J3 fusions

  • Bimolecular Fluorescence Complementation (BiFC)

  • Validation with co-localization using OR10J3 antibodies (1:100 dilution of fluorophore-conjugated versions)

• Mass spectrometry-coupled approaches:

  • Antibody-purified OR10J3 complexes analyzed by LC-MS/MS

  • SILAC or TMT labeling for quantitative interactomics

  • Comparison between stimulated and unstimulated conditions

• Heterologous expression systems:

  • HEK293 cells for controlled expression

  • Baculovirus-infected insect cells for higher yields

  • Verification of expression using Western blot with OR10J3 antibodies at 1:1000 dilution

• Functional validation of interactions:

  • siRNA knockdown of putative interactors followed by OR10J3 localization studies

  • Competitive peptide inhibition of specific interactions

  • FRET/FLIM analysis between OR10J3 and putative partners

These methods capitalize on the specificity of OR10J3 antibodies to identify the G proteins, trafficking adaptors, and regulatory proteins that control OR10J3 function and localization .

How can OR10J3 antibodies contribute to understanding the molecular basis of olfactory receptor specificity?

OR10J3 antibodies can be leveraged to elucidate molecular mechanisms of olfactory receptor specificity through these advanced approaches:

• Structural studies with antibody-based stabilization:

  • Antibody-mediated crystallization of OR10J3

  • Single-particle cryo-EM with antibody fragments

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) with and without ligands

  • Surface plasmon resonance with immobilized antibody and OR10J3 complexes

• Ligand-induced conformational changes:

  • Differential epitope exposure assays upon ligand binding

  • FRET-based sensors using strategically placed antibody-fluorophore conjugates

  • Protease protection assays with antibody detection of fragments

  • Use of 1:200 dilution of fluorophore-conjugated antibodies for detection

• Phage display-derived affinity reagents for OR10J3:

  • Selection of sequence-specific and conformation-specific antibodies

  • Machine learning approaches for predicting binding modes from selection data

  • Design of antibodies with customized specificity profiles

  • Computational disentangling of binding modes associated with different ligands

• Spatial organization studies:

  • Super-resolution imaging of OR10J3 nanoclusters

  • Quantification of receptor oligomerization states

  • Co-localization with lipid raft markers

  • Detection protocols using 1:50-1:100 conjugated antibodies

• Comparative studies across olfactory receptor subtypes:

  • Epitope mapping to conserved vs. variable regions

  • Cross-reactivity analysis with related olfactory receptors

  • Evolutionary conservation of binding sites

• Functional correlation studies:

  • Calcium imaging following antibody modulation of receptor function

  • Electrophysiological recording with antibody perfusion

  • cAMP accumulation assays with epitope-specific antibodies

These approaches leverage OR10J3 antibodies not only as detection tools but as modulators of receptor function and conformation, providing insights into the molecular basis of olfactory discrimination .

How do OR10J3 expression patterns compare across different tissue types and developmental stages?

Comprehensive analysis of OR10J3 expression across tissues and development reveals distinct patterns that can be investigated using specialized immunohistochemical approaches:

• Tissue-specific expression profiles:

• Developmental regulation:

  • Embryonic expression begins at neurogenesis of olfactory placode

  • Progressive increase through fetal development

  • Peak expression in early postnatal period

  • Maintenance at stable levels throughout adulthood

  • Use of 1:50 dilution of Alexa Fluor conjugates recommended for developmental studies

• Methodological considerations for developmental studies:

  • Age-appropriate fixation protocols (shorter times for embryonic tissues)

  • Specialized antigen retrieval for developmental tissues

  • Controls for each developmental stage

  • Quantitative image analysis with standardized exposure settings

• Comparative species analysis:

  • Human and rat show highest antibody cross-reactivity

  • Species-specific optimization may be required

  • Evolutionary conservation of expression patterns can be assessed

• Single-cell resolution approaches:

  • Combination with cell-type specific markers

  • Correlation with single-cell RNA-seq data

  • Spatial transcriptomics validation

These comparative analyses reveal that while OR10J3 expression is primarily associated with olfactory sensory neurons, it also shows unexpected expression in certain non-neuronal contexts, suggesting potential roles beyond classical olfaction .

What are the best approaches for multiplex detection of OR10J3 alongside other olfactory signaling components?

Multiplexed detection of OR10J3 with other components of the olfactory signaling pathway requires careful experimental design and antibody selection:

• Antibody panel design considerations:

• Sequential multiplexing protocols:

  • Tyramide signal amplification (TSA) with sequential antibody stripping

  • Zenon labeling technology for same-species antibodies

  • Primary antibody directly conjugated to distinct fluorophores

  • Spectral unmixing for overlapping fluorophores

• Alternative multiplexing approaches:

  • Imaging Mass Cytometry (IMC) with metal-conjugated antibodies

  • Cyclic immunofluorescence (cycIF)

  • DNA-barcoded antibodies with sequential readout

  • Multi-epitope ligand cartography (MELC)

• Spatial context preservation:

  • Thick-section (40-100 μm) immunostaining with confocal analysis

  • Tissue clearing techniques (CLARITY, iDISCO) compatible with immunolabeling

  • 3D reconstruction of complete olfactory circuits

• Analysis and quantification:

  • Colocalization coefficients (Pearson's, Manders')

  • Proximity analysis (nearest neighbor distances)

  • Spatial statistics (Ripley's K-function, pair correlation)

These approaches enable comprehensive mapping of the OR10J3 signaling complex within its native cellular context, providing insights into the spatial organization of olfactory signal transduction .

How can systems biology approaches integrate OR10J3 antibody-derived data with other omics datasets?

Integrating antibody-derived data on OR10J3 with multi-omics datasets enables systems-level understanding of olfactory receptor function:

• Multi-modal data integration frameworks:

  • Correlation of protein abundance (antibody-based) with transcriptomic data

  • Integration with phosphoproteomics for signaling network reconstruction

  • Metabolomic correlation with receptor activation states

  • Chromatin accessibility data to understand epigenetic regulation

• Network modeling approaches:

  • Protein-protein interaction networks centered on OR10J3

  • Signaling pathway reconstruction from phosphoproteomic data

  • Dynamic network analysis of temporal activation patterns

  • Bayesian network inference incorporating antibody-derived quantification

• Single-cell multi-omic integration:

  • CITE-seq with OR10J3 antibodies for simultaneous protein and RNA quantification

  • Spatial proteomics correlated with spatial transcriptomics

  • Trajectory analysis of olfactory neuron differentiation

  • Clonal evolution studies with lineage tracing

• Mathematical modeling applications:

  • Kinetic models of OR10J3 signaling incorporating antibody-derived quantitative data

  • Stochastic models of olfactory receptor expression

  • Information theory applications to receptor coding

  • Machine learning models leveraging biophysical constraints

• Visualization and analysis platforms:

  • Cytoscape extensions for multi-omic data visualization

  • R Bioconductor packages for integrated analysis

  • Interactive dashboards for exploring relationships between datasets

  • 3D spatial visualization of protein localization data

• Practical implementation considerations:

  • Data normalization across platforms

  • Batch effect correction methods

  • Missing value imputation strategies

  • Metadata standardization and ontology mapping

This systems biology approach transforms antibody-derived data from descriptive observations to predictive models of OR10J3 function within the broader context of olfactory perception, potentially revealing emergent properties not evident from single-method studies .