OR10J1 Antibody

What is OR10J1 and why is it significant in research?

OR10J1 (Olfactory Receptor 10J1) is a G-protein-coupled receptor (GPCR) originally identified in olfactory sensory neurons. It belongs to the large family of olfactory receptors responsible for the recognition and G protein-mediated transduction of odorant signals . Recent research has revealed that olfactory receptors, including OR10J1, are expressed in tissues beyond the olfactory epithelium and may have significant functions in immune responses, cell-cell recognition, migration, and proliferation .

The significance of OR10J1 in research has grown as studies have demonstrated its expression in human spermatozoa and potential roles in various physiological processes outside of olfaction. Investigating OR10J1 can provide insights into novel signaling mechanisms in non-neuronal tissues and their implications in various disease processes.

What are the key specifications of commercially available OR10J1 antibodies?

OR10J1 antibodies are available in various formats with different specifications as summarized in the table below:

Most commercially available antibodies are affinity-purified from rabbit antiserum using epitope-specific immunogen chromatography techniques .

How is OR10J1 protein characterized structurally and functionally?

OR10J1 is characterized as a 7-transmembrane domain protein typical of the G-protein-coupled receptor superfamily . Structurally, it shares homology with other olfactory receptors and contains the conserved domains necessary for G-protein signal transduction.

Key functional and structural characteristics include:

• UniProt Primary Accession: P30954 (with secondary accessions Q2M1M8, Q5VSV1, Q6IET5, Q96R56)

• Molecular weight: Approximately 35 kDa

• Cellular localization: Cell membrane; multi-pass membrane protein

• Tissue specificity: Originally identified in olfactory neurons, but also detected in cerebellum and testis

• Function: Primarily acts as an odorant receptor, binding specific molecules to initiate G-protein signaling cascades

• Protein family: Belongs to the G-protein coupled receptor 1 family

Research has expanded our understanding of OR10J1 beyond its classical role in olfaction, suggesting potential functions in other physiological processes and signaling pathways.

How can OR10J1 antibodies be optimized for immunodetection in non-neuronal tissues?

Optimizing OR10J1 antibody use for non-neuronal tissues requires careful consideration of several methodological factors:

Western Blot Optimization:

• Protein extraction: Use specialized membrane protein extraction protocols with non-ionic detergents (e.g., Triton X-100, NP-40) to efficiently solubilize membrane-bound OR10J1

• Sample preparation: Heat samples at 37°C instead of boiling to prevent GPCR aggregation

• Blocking: 5% non-fat milk or 3-5% BSA in TBST, with extended blocking times (2-3 hours)

• Antibody dilution: Start with manufacturer's recommendation (typically 1:500-1:1000 for WB) and optimize as needed

• Incubation conditions: 4°C overnight for primary antibody binding

• Controls: Include positive control (e.g., Jurkat cells, which have been demonstrated to express OR10J1)

Immunofluorescence Protocol Refinement:

• Fixation: 4% paraformaldehyde (10-15 minutes) followed by permeabilization with 0.1% Triton X-100

• Autofluorescence reduction: Treat with 0.1% sodium borohydride

• Blocking: PBS with 5% normal goat serum and 1% fish gelatin

• Antibody dilution: Typically 1:200-1:1000

• Incubation: Overnight at 4°C for primary antibody

• Nuclear counterstain: DAPI

• Mounting: Use anti-fade mounting medium to preserve fluorescence

Evidence from research indicates that OR10J1 shows punctate staining patterns in certain cell types, such as in the flagella of human spermatozoa, which should be considered when interpreting immunofluorescence results .

What experimental approaches can be used to investigate OR10J1 function in immune responses?

Recent research has revealed potential roles for olfactory receptors in immune function, particularly in the context of infections such as malaria . To investigate OR10J1's potential function in immune responses, consider these approaches:

1. Expression Analysis in Immune Cells:

• qRT-PCR to quantify OR10J1 expression in various immune cell populations

• Single-cell RNA-Seq to identify specific immune cell subsets expressing OR10J1

• Western blot and flow cytometry to confirm protein expression

2. Functional Characterization:

• CRISPR/Cas9-mediated knockout of OR10J1 in immune cell lines

• Overexpression studies using lentiviral vectors

• Phosphorylation studies of downstream signaling molecules (TBK1, IRF3, P38, JNK)

• NF-κB and IFN-β promoter reporter assays to assess impact on inflammatory signaling

3. Infection Models:

• In vitro infection models using pathogens shown to modulate olfactory receptor expression

• Ex vivo analysis of OR10J1 expression in patient samples during infection

• Animal models with conditional OR10J1 knockout in specific immune cell populations

4. Ligand Identification:

• Screening of pathogen metabolites as potential OR10J1 ligands

• Using calcium flux assays to measure receptor activation

• G-protein signaling assays to assess downstream functional consequences

Recent research on olfactory receptor Olfr1386 demonstrated its role in regulating type I interferon responses during malaria infection, with NAD identified as a potential ligand . Similar approaches could be applied to investigate OR10J1's role in immune function.

What are the current challenges in studying OR10J1 localization and expression in different tissue types?

Investigating OR10J1 across diverse tissues presents several challenges that require methodological solutions:

1. Low Expression Levels:

• Challenge: ORs typically have low expression outside olfactory tissue

• Solution: Use highly sensitive detection methods such as RNAscope, droplet digital PCR, or nested PCR approaches

• Evidence: Studies detecting OR expression in human spermatozoa required RNA-Seq approaches to identify transcripts

2. Antibody Specificity:

• Challenge: High sequence homology between different ORs can lead to cross-reactivity

• Solution: Validate antibody specificity using OR10J1-overexpressing cells versus knockout controls

• Methodology: Recombinant expression systems such as Hana3A cells (specialized for OR expression) can be used for validation

3. Sub-cellular Localization:

• Challenge: Determining precise localization of OR10J1 in different cell types

• Solution: Super-resolution microscopy techniques (STORM, PALM) combined with subcellular fractionation

• Evidence: OR10J1 has been detected with weak punctate staining in the flagella of sperm, with stronger staining 3μm caudal to the midpiece

4. Functional Expression:

• Challenge: Ensuring proper trafficking and functionality in heterologous expression systems

• Solution: Co-expression with receptor trafficking proteins (e.g., RTP1S) and specific Gα subunits (e.g., Gαolf)

• Methodology: Calcium imaging assays to confirm functional expression

5. Tissue Preparation:

• Challenge: Preserving membrane protein integrity during processing

• Solution: Optimized fixation protocols (e.g., shorter fixation times, specialized fixatives for GPCRs)

• Methodology: Use membrane-preserving fixatives like periodate-lysine-paraformaldehyde for immunohistochemistry

How has OR10J1 been implicated in human spermatozoa function?

Research has identified OR10J1 as one of several olfactory receptors expressed in human spermatozoa, suggesting potential roles in sperm function and fertility:

Expression Pattern:

• RNA-Seq analysis has detected OR10J1 transcripts in human spermatozoa samples

• Immunofluorescence studies using α-OR10J1 antibodies revealed a distinctive staining pattern: weak punctate staining in the flagella with stronger punctate staining approximately 3μm caudal to the midpiece

Potential Functional Roles:

• Chemotaxis/chemokinesis: ORs may help sperm navigate the female reproductive tract by detecting chemical cues

• Capacitation: Potential involvement in sperm maturation processes required for fertilization

• Acrosome reaction: May respond to zona pellucida or follicular fluid components

Methodological Approaches Used:

• RNA isolation from purified motile spermatozoa populations

• RNA-Seq analysis of spermatozoal transcriptome

• Validation of expression using strand-specific mRNA-Seq

• Immunofluorescence localization using specific α-OR10J1 antibodies

• Heterologous expression systems (Hana3A cells) for functional validation

This research area represents an emerging field in reproductive biology, with OR10J1 potentially serving as a therapeutic target for fertility treatments or contraceptives in the future.

What is the significance of OR10J1 in malaria and other infectious disease research?

While OR10J1 itself has not been directly studied in malaria research, recent findings regarding other olfactory receptors in this context suggest potential significance:

Key Research Findings:

• Multiple olfactory receptor genes are expressed in mouse spleen during malaria parasite infections

• Human blood samples from malaria patients show altered expression of 42 different OR genes

• The olfactory receptor Olfr1386 regulates type I interferon responses during Plasmodium yoelii infection

• Parasite RNA can increase expression of certain olfactory receptors

• NAD (nicotinamide adenine dinucleotide) has been identified as a potential ligand for Olfr1386, activating immune signaling pathways

Potential Roles of ORs in Infection:

• Sensing of pathogen-derived metabolites

• Modulation of host immune responses

• Regulation of inflammatory signaling pathways

• Integration with toll-like receptor signaling

Research Implications for OR10J1:

• OR10J1 might play similar roles in sensing infection-associated molecules

• Could be involved in early detection of pathogen presence

• May modulate specific immune response pathways

• Could represent a novel therapeutic target for infectious diseases

This emerging research area suggests that olfactory receptors, potentially including OR10J1, may have previously unrecognized roles in immune function during infectious disease, opening new avenues for therapeutic intervention.

What methodological approaches are recommended for OR10J1 knockdown or knockout studies?

For researchers investigating OR10J1 function through gene silencing or deletion, several methodological approaches are recommended:

CRISPR/Cas9 Gene Editing:

• Guide RNA design: Target conserved regions of OR10J1 using multiple bioinformatic tools to minimize off-target effects

• Verification strategy: Design PCR primers spanning the targeted region for deletion confirmation

• Cell delivery: Use nucleofection for primary cells or lipofection/viral transduction for established cell lines

• Validation: Confirm knockout through sequencing, qRT-PCR, and western blotting

• Controls: Include both wild-type and CRISPR non-targeting controls

siRNA/shRNA Knockdown:

• siRNA design: Target regions with minimal homology to other OR family members

• Delivery method: Lipid-based transfection for cell lines; electroporation for primary cells

• Validation: Confirm knockdown efficiency by qRT-PCR (>70% reduction recommended)

• Timing: Assess effects 48-72 hours post-transfection for transient knockdown

• Controls: Include scrambled siRNA/shRNA controls

Functional Validation:

• Phenotypic assays: Design based on hypothesized OR10J1 function (calcium imaging, migration assays, etc.)

• Rescue experiments: Re-express OR10J1 in knockout cells to confirm specificity

• Pathway analysis: Assess changes in downstream signaling molecules (e.g., phosphorylation of TBK1, IRF3, P38, JNK)

• Promoter reporter assays: Use IFN-β and NF-κB promoter-driven luciferase constructs to assess impact on signaling

Considerations from Recent Research:
Recent studies with olfactory receptor Olfr1386 knockout mice showed significantly lower IFN-α/β production during malaria infection . Similar approaches could be applied to OR10J1 research, with careful consideration of potential compensation by other olfactory receptor family members.

How can researchers address common issues with western blot detection of OR10J1?

Western blot detection of GPCRs like OR10J1 can be challenging due to their hydrophobic nature and multiple transmembrane domains. Here are solutions to common issues:

Weak or No Signal:

• Protein extraction: Use specialized GPCR extraction buffers containing 1-2% non-ionic detergents (Triton X-100, NP-40, or DDM)

• Sample preparation: Avoid boiling samples (use 37°C for 5-10 minutes instead)

• Antibody concentration: Increase primary antibody concentration (try 1:500 instead of 1:1000)

• Incubation time: Extend primary antibody incubation to overnight at 4°C

• Detection system: Switch to more sensitive detection methods (e.g., enhanced chemiluminescence plus)

Multiple Bands/Non-specific Bands:

• Blocking: Increase blocking time (3-4 hours) and concentration (5% BSA)

• Washing: Extend wash steps (5-6 washes of 10 minutes each)

• Antibody specificity: Validate with positive controls (e.g., Jurkat cells for OR10J1)

• Competition assay: Pre-incubate antibody with immunizing peptide to confirm specificity

• Sample purity: Ensure membrane fractions are properly prepared to reduce cytosolic contamination

Band Size Discrepancy:

• Post-translational modifications: GPCRs often show different apparent molecular weights due to glycosylation

• Sample preparation: Treat with deglycosylation enzymes (PNGase F) to verify core protein size

• Aggregation: Add urea (up to 4M) to disrupt GPCR aggregates that may appear as higher molecular weight bands

• Degradation: Add additional protease inhibitors to prevent proteolytic cleavage

Optimization Strategy:
Start with the manufacturer's recommended protocol, then systematically modify one variable at a time while keeping others constant. Document all changes and results to determine optimal conditions for your specific experimental system.

What are the best approaches for validating OR10J1 antibody specificity?

Validating antibody specificity is crucial for reliable research with OR10J1. Here are comprehensive approaches:

Positive Controls:

• Overexpression systems: Use HEK293T or Hana3A cells (specialized for OR expression) transfected with OR10J1 expression plasmids

• Epitope tagging: Co-express OR10J1 with N-terminal tags (e.g., rhodopsin tag/rho-tag) that can be detected with established antibodies

• Known expressing tissues: Test antibody on tissues with confirmed OR10J1 expression (e.g., Jurkat cells)

Negative Controls:

• Knockout/knockdown validation: Test antibody on CRISPR-generated OR10J1 knockout cells or siRNA-treated samples

• Peptide competition: Pre-incubate antibody with the immunizing peptide to block specific binding

• Secondary-only controls: Perform staining with secondary antibody alone to identify non-specific binding

Cross-Reactivity Assessment:

• Sequence homology analysis: Identify ORs with high homology to OR10J1 in the immunogen region

• Heterologous expression: Test antibody on cells expressing closely related ORs

• Multiple antibody comparison: Test different antibodies targeting distinct epitopes of OR10J1

Technical Validation Approaches:

• Western blot: Confirm single band at expected molecular weight (~35 kDa)

• Immunoprecipitation followed by mass spectrometry: Definitively identify pulled-down proteins

• Microscopy validation: Confirm expected subcellular localization (membrane staining for OR10J1)

• Multi-method confirmation: Validate findings using orthogonal techniques (IF, WB, flow cytometry)

Research has shown that OR antibody validation is particularly important due to the large number of OR genes with similar sequences. The most rigorous validation employs both overexpression and knockout/knockdown approaches.

How can researchers optimize immunofluorescence protocols for detecting OR10J1 in fixed tissues?

Detecting OR10J1 in fixed tissues requires optimized protocols to overcome challenges associated with membrane protein detection:

Fixation Optimization:

• Fixative selection: 4% paraformaldehyde (10-15 minutes) preserves membrane protein epitopes better than alcoholic fixatives

• Mild fixation: For sensitive epitopes, reduce fixation time or concentration (2% PFA for 5-10 minutes)

• Alternative fixatives: Test methanol-free formaldehyde or periodate-lysine-paraformaldehyde for improved membrane protein preservation

• Post-fixation: Add a brief post-fixation step with 0.1% glutaraldehyde to better preserve membrane structure

Antigen Retrieval Methods:

• Heat-induced epitope retrieval: Citrate buffer (pH 6.0) at 95-98°C for 15-20 minutes

• Enzymatic retrieval: Proteinase K (1-5 μg/ml for 5-15 minutes) can expose membrane epitopes

• Detergent permeabilization: Optimize Triton X-100 concentration (0.1-0.3%) and time

• SDS treatment: Brief treatment with 1% SDS can improve detection of some membrane proteins

Signal Amplification:

• Tyramide signal amplification: Enhances detection of low-abundance proteins

• Polymer detection systems: HRP-polymer conjugates provide increased sensitivity

• Fluorescent secondary antibodies: Use highly cross-adsorbed versions to reduce background

• Extended primary antibody incubation: Overnight at 4°C with optimized dilution (1:200-1:1000)

Background Reduction:

• Extended blocking: Use 5% normal serum + 1% fish gelatin to reduce non-specific binding

• Autofluorescence reduction: Treat with sodium borohydride (0.1% for 10 minutes) or Sudan Black B

• Tissue pre-treatment: Block endogenous peroxidases and biotin if using amplification systems

• Multiple washing steps: Increase number and duration of washes with 0.1% Tween-20 in PBS

Expected Staining Pattern:
Based on research, OR10J1 shows punctate staining patterns in certain tissues, such as in human spermatozoa, where it displays weak punctate staining in the flagella with stronger staining 3μm caudal to the midpiece . This pattern can guide interpretation of results.

What are emerging research areas concerning OR10J1 in non-olfactory functions?

Recent discoveries about olfactory receptors in non-olfactory tissues open several promising research directions for OR10J1:

Immune System Regulation:

• Investigating OR10J1 expression changes during infection and inflammation

• Exploring potential roles in specific immune cell populations (similar to Olfr1386 in malaria response)

• Examining interactions with pattern recognition receptor pathways

• Studying OR10J1 responses to pathogen-derived metabolites

Reproductive Biology:

• Further characterizing OR10J1 function in human spermatozoa

• Investigating potential chemosensory roles in sperm navigation

• Exploring connections to male fertility disorders

• Developing potential contraceptive approaches targeting OR10J1

Cell Signaling Pathways:

• Mapping OR10J1-activated signaling cascades beyond canonical G-protein pathways

• Investigating cross-talk with cytokine signaling networks

• Studying roles in calcium flux, cAMP modulation, and MAPK pathway activation

• Exploring interactions with TBK1, IRF3, P38, and JNK phosphorylation pathways

Disease Associations:

• Examining OR10J1 expression changes in disease states

• Investigating potential roles in inflammatory disorders

• Exploring associations with reproductive pathologies

• Analyzing genetic variants affecting OR10J1 function

Therapeutic Applications:

• Development of specific OR10J1 agonists/antagonists

• Exploring OR10J1 as a drug target for infectious disease

• Investigating applications in reproductive medicine

• Considering diagnostic potential of OR10J1 expression patterns

The discovery that nicotinamide adenine dinucleotide (NAD) acts as a ligand for another olfactory receptor suggests that OR10J1 might similarly respond to endogenous metabolites, opening new avenues for investigation into its physiological functions beyond olfaction.

How might developments in GPCR structural biology advance OR10J1 research?

Recent advances in GPCR structural biology offer significant opportunities for OR10J1 research:

Cryo-EM Applications:

• Single-particle cryo-EM now achieves resolution sufficient for GPCR structure determination

• Potential to resolve OR10J1 structure in various conformational states

• Opportunity to visualize OR10J1 in complex with G-proteins and other signaling partners

• Requires optimization of expression and purification protocols specific for olfactory receptors

Computational Approaches:

• Homology modeling based on resolved GPCR structures

• Molecular dynamics simulations to predict ligand binding and conformational changes

• AI-based approaches (AlphaFold2) for structure prediction with increasing accuracy

• Virtual screening of potential ligands against predicted binding pockets

Ligand Discovery Platforms:

• High-throughput screening using biosensor technologies

• Fragment-based drug discovery applied to olfactory receptors

• Chemoinformatics approaches to identify potential endogenous ligands

• Development of OR10J1-specific nanobodies as crystallization chaperones

Methodological Innovations:

• Lipid cubic phase crystallization optimized for olfactory receptors

• Nanodiscs and styrene maleic acid lipid particles (SMALPs) for stabilization

• Serial femtosecond crystallography at X-ray free electron lasers

• Integration of hydrogen-deuterium exchange mass spectrometry for dynamics information

Translational Impact:

• Structure-based design of OR10J1 modulators for research and therapeutic applications

• Enhanced understanding of ligand selectivity across the olfactory receptor family

• Deeper insights into GPCR evolution and functional diversification

• Development of biosensors based on engineered OR10J1 variants

These advances could transform OR10J1 research by providing atomic-level insights into its function, facilitating the rational design of modulators, and enhancing our understanding of its physiological roles.

What interdisciplinary approaches might yield new insights into OR10J1 function?

Interdisciplinary research approaches offer powerful strategies for uncovering novel aspects of OR10J1 biology:

Systems Biology Integration:

• Multi-omics analysis to correlate OR10J1 expression with metabolomic, proteomic, and transcriptomic changes

• Network analysis to identify functional relationships with other signaling pathways

• Mathematical modeling of OR10J1 signaling dynamics in different cellular contexts

• Pathway enrichment analysis to identify biological processes associated with OR10J1 activity

Bioengineering Approaches:

• CRISPR-based genetic screens to identify functional partners and modulators

• Development of OR10J1 biosensors for real-time activity monitoring

• Optogenetic control of OR10J1 signaling for precise temporal studies

• Synthetic biology circuits incorporating OR10J1 for novel sensing applications

Advanced Imaging Technologies:

• Super-resolution microscopy to visualize OR10J1 nanoclusters and interactions

• FRET/BRET assays to monitor protein-protein interactions in real-time

• Live-cell imaging with genetically encoded sensors to track signaling events

• Correlative light and electron microscopy for ultrastructural localization

Translational Research Connections:

• Patient-derived organoids to study OR10J1 in disease-relevant contexts

• Single-cell analysis of OR10J1 expression in clinical samples

• Genome-wide association studies to identify OR10J1 variants linked to disease

• Development of humanized mouse models for OR10J1 functional studies

Evolutionary Biology Perspectives:

• Comparative genomics to understand OR10J1 conservation and divergence

• Analysis of selection pressures on OR10J1 across species

• Reconstruction of ancestral OR sequences to understand functional evolution

• Ecological connections between environmental chemicals and OR10J1 evolution

By combining these interdisciplinary approaches, researchers can develop a more comprehensive understanding of OR10J1's diverse functions beyond classical olfaction, potentially revealing novel therapeutic targets and biological mechanisms.