OR14J1 Antibody

What is OR14J1 and why is it studied in research?

OR14J1 (Olfactory Receptor Family 14 Subfamily J Member 1) is a G protein-coupled receptor functioning as an odorant receptor. Beyond its canonical role in olfaction, recent research has identified its expression in human spermatozoa, suggesting non-olfactory functions . OR14J1 is also known by alternative designations including Hs6m1-28, Olfactory Receptor 5U1, and Olfactory Receptor Or6-25 . It is a multi-pass membrane protein (Gene ID: 442191, UniProt ID: Q9UGF5) that has gained research interest for understanding the broader physiological roles of olfactory receptors outside the nasal epithelium .

What types of OR14J1 antibodies are available for research applications?

Multiple OR14J1 antibodies are available with varying specifications:

Each antibody is generated from rabbits immunized with KLH-conjugated synthetic peptides corresponding to specific regions of the human OR14J1 protein .

What are the optimal applications for OR14J1 antibodies?

OR14J1 antibodies can be employed in multiple applications, with performance varying by conjugation and epitope:

• Western Blotting (WB): Most unconjugated OR14J1 antibodies and some conjugated versions (FITC, Biotin) are suitable for WB .

• Immunohistochemistry (IHC): Both paraffin-embedded (IHC-p) and general IHC applications are supported .

• ELISA: Most conjugated antibodies perform well in ELISA applications with recommended dilutions around 1:5000 .

• Immunofluorescence (IF/ICC): Specific antibodies like STJ94784 are optimized for IF with recommended dilutions of 1:200-1:1000 .

For optimal results, validation with positive and negative controls is essential, particularly when studying tissues with potential cross-reactivity to other olfactory receptors.

What methodological approaches can be used to study OR14J1 expression in human spermatozoa?

A multi-technique approach is recommended:

• RNA-Seq analysis: For transcript detection and quantification, RNA-Seq has been successfully used to characterize OR expression in human spermatozoa. This revealed both sense and antisense transcripts for many ORs including OR14J1-related ORs .

• Immunolocalization: Using validated OR14J1 antibodies for immunofluorescence microscopy on fixed spermatozoa (recommended dilution 1:200-1:1000) .

• Protein validation: Western blotting to confirm protein expression and size, typically using unconjugated antibodies at manufacturer-recommended dilutions .

• Functional studies: For investigating physiological roles, calcium imaging in heterologous expression systems has been used for other ORs and could be applied to OR14J1 .

Researchers should be aware that both transcripts and proteins may show individual variations among sperm donors, as demonstrated in comprehensive RNA-Seq analyses .

How should I design experiments to validate OR14J1 antibody specificity?

A comprehensive validation strategy includes:

• Positive controls: Transfect Hana3A cells (or similar expression systems) with OR14J1 expression vectors. This approach has been successful for other ORs, using plasmids containing the complete OR open reading frame with tags (e.g., rhodopsin tag) to facilitate detection .

• Negative controls: Include untransfected cells and cells expressing related ORs to assess cross-reactivity.

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide before immunostaining to confirm specificity.

• Multiple antibody comparison: Use antibodies targeting different epitopes of OR14J1 (e.g., AA 206-234 vs. AA 272-321) to confirm consistent localization patterns .

• Genetic approaches: When possible, use genetic knockout or knockdown models to confirm signal absence in tissues naturally expressing OR14J1.

What are the optimal fixation and immunostaining protocols for OR14J1 detection?

Based on protocols used for other olfactory receptors:

• Cell fixation: Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature.

• Permeabilization: Use PBS containing 0.1% Triton X-100 for membrane permeabilization.

• Blocking: Block with PBS containing 0.1% Triton X-100, 5% normal goat serum, and 1% fish gelatin for 1 hour at room temperature .

• Primary antibody incubation: Apply diluted OR14J1 antibody (typical ranges: 1:50-1:200 for unconjugated; 1:200-1:1000 for IF applications) and incubate overnight at 4°C .

• Secondary antibody: For unconjugated primary antibodies, use appropriate Alexa-conjugated secondary antibodies with DAPI counterstain, incubating for 45 minutes at room temperature protected from light .

For conjugated antibodies (FITC, etc.), adjust protocols accordingly and validate signal specificity with appropriate controls.

How does OR14J1 expression and function compare to other olfactory receptors in non-olfactory tissues?

While the search results don't provide specific information about OR14J1 function, research on related ORs provides context:

• Expression patterns: Comprehensive RNA-Seq analysis has revealed that numerous ORs, including those related to OR14J1, are expressed in human spermatozoa. Some ORs show higher expression in sperm than in any other tissue investigated, even exceeding expression of housekeeping genes like TBP and GUSB .

• Antisense transcripts: Many ORs exhibit both sense and antisense transcripts in spermatozoa. These antisense transcripts may have regulatory functions or represent novel non-coding RNAs. Whether OR14J1 specifically has antisense transcripts would require further investigation .

• Functional roles: Some ORs, such as OR1D2, OR7A5, and OR4D1, have been characterized in human spermatozoa, with potential roles in sperm chemotaxis or other reproductive functions. OR14J1's specific function remains to be elucidated but may parallel these other receptors .

Understanding OR14J1 in this broader context can help researchers design experiments to investigate its specific roles in reproductive or other non-olfactory tissues.

What approaches can be used to investigate potential ligands for OR14J1?

Although the search results don't specifically address OR14J1 ligand discovery, established methods for OR deorphanization can be applied:

• Heterologous expression systems: Transfect Hana3A cells (which express necessary accessory proteins) with OR14J1 expression constructs alongside signaling components like Gαolf .

• Calcium imaging assays: After expression in appropriate cell lines, potential ligands can be screened using calcium-sensitive dyes to detect receptor activation.

• Plasmid construction: Amplify the OR14J1 coding sequence from human genomic DNA using PCR with specific primers, then subclone into expression vectors (e.g., pCI) containing an N-terminal tag (like rhodopsin tag) to improve surface expression .

• Accessory protein co-expression: Include RTP1S and other trafficking proteins to enhance functional expression at the cell surface .

• Compound library screening: Test candidate odorants or tissue-specific metabolites in a high-throughput manner to identify potential physiological ligands.

What are common challenges when working with OR14J1 antibodies and how can they be addressed?

Based on experiences with other OR antibodies:

• Background signal: ORs often have sequence similarities, leading to potential cross-reactivity. Solutions include:

  • Increasing antibody dilution (try ranges from 1:200-1:1000 for IF applications)

  • More stringent blocking (use 5% normal serum + 1% fish gelatin)

  • Pre-absorption with related peptides

• Poor signal strength: ORs are often expressed at low levels. Consider:

  • Signal amplification techniques

  • Using antibodies against specific epitope regions known to be accessible

  • Optimizing fixation protocols to preserve epitope integrity

• Inconsistent results: Given the variability in OR expression between individuals , use:

  • Pooled samples when appropriate

  • Increased biological replicates

  • Standardized positive controls (e.g., transfected cell lines)

• Storage and handling: Store antibodies at -20°C for up to 1 year and avoid repeated freeze-thaw cycles to maintain activity .

How can I distinguish between closely related olfactory receptors when using antibodies?

This requires careful experimental design:

• Epitope selection: Choose antibodies targeting unique regions of OR14J1. The C-terminal region (where many available antibodies target) often offers greater sequence divergence between ORs .

• Western blot validation: Confirm antibody specificity by expressing individual ORs with epitope tags in heterologous systems.

• Comparative analysis: Use multiple antibodies targeting different epitopes of OR14J1 to verify consistent results.

• Complementary methods: Combine antibody-based detection with nucleic acid-based methods like RNA-Seq or RT-PCR to corroborate protein findings with transcript data .

• Control experiments: Include peptide competition assays and validation in tissues known to express or lack OR14J1 to confirm specificity.