OR1K1 Antibody

What is OR1K1 and why is it significant in olfactory research?

OR1K1 (Olfactory Receptor 1K1) is a protein encoded by the OR1K1 gene in humans, belonging to the G-protein coupled receptor 1 family . This receptor functions as an odorant receptor that interacts with odorant molecules in the nose to initiate neuronal responses triggering smell perception . OR1K1 features a 7-transmembrane domain structure shared with many neurotransmitter and hormone receptors, participating in G protein-mediated transduction of odorant signals .

The significance of OR1K1 in olfactory research stems from its role in the fundamental mechanisms of olfactory perception. The olfactory receptor gene family, to which OR1K1 belongs, is the largest gene family in the human genome . OR1K1 is located on Chromosome 9 and contributes to our understanding of how odor information is encoded and transmitted to the brain .

What are the key specifications of commercially available OR1K1 antibodies?

Commercial OR1K1 antibodies are designed to detect endogenous levels of OR1K1 protein in experimental settings . The following table summarizes key specifications of a typical OR1K1 polyclonal antibody:

These specifications ensure researchers can select appropriate experimental conditions when working with OR1K1 antibodies .

What is the optimal protocol for OR1K1 antibody validation?

A methodological approach to validating OR1K1 antibody specificity requires multiple complementary techniques:

• Blocking peptide experiments: Pre-incubate the antibody with the immunizing peptide (derived from human OR1K1) to confirm signal abolishment in subsequent detection methods .

• Genetic validation: Test antibody performance in OR1K1 knockout/knockdown models. Signal absence in these models strongly supports specificity .

• Expression system testing: Compare antibody detection between OR1K1-transfected cells and control cells. Transfection protocols should use expression vectors (such as pME18S) containing the OR1K1 gene, as demonstrated in functional analysis studies .

• Western blot band analysis: Confirm that the detected band appears at the expected molecular weight (34 kD for OR1K1) .

• Cross-reactivity assessment: Test against closely related olfactory receptors to ensure the antibody does not detect other family members.

• Multiple application validation: Confirm consistent results across different experimental platforms (WB, ELISA, immunocytochemistry).

This comprehensive validation approach ensures reliable experimental outcomes in OR1K1 research .

What are the recommended storage and handling practices for OR1K1 antibodies?

Proper storage and handling of OR1K1 antibodies are essential for maintaining antibody performance and experimental reproducibility:

• Long-term storage: Store antibody at -20°C, where it maintains stability for approximately one year .

• Formulation considerations: OR1K1 antibodies are typically supplied in PBS containing 50% glycerol and 0.02% sodium azide, which helps maintain stability .

• Avoid freeze-thaw cycles: Repeated freezing and thawing significantly degrades antibody performance. The product literature specifically warns against this practice .

• Aliquoting strategy: Upon receipt, divide the antibody into small single-use aliquots before freezing to minimize freeze-thaw cycles.

• Short-term storage: For antibodies in active use, store at 4°C for up to two weeks.

• Transportation conditions: Maintain cold chain during transport, using ice packs or dry ice as appropriate.

• Contamination prevention: Use sterile technique when handling antibody solutions to prevent microbial contamination.

Following these methodological guidelines ensures optimal antibody performance throughout the research project timeline .

How can OR1K1 antibody be integrated into studies of olfactory signal transduction?

The integration of OR1K1 antibody into olfactory signal transduction research requires sophisticated methodological approaches:

• Co-immunoprecipitation studies: OR1K1 antibody can be used to pull down receptor complexes, identifying interacting partners in the signaling cascade. This approach helps map protein-protein interactions involved in OR1K1-mediated signaling .

• Subcellular localization analysis: Immunocytochemistry using OR1K1 antibody can track receptor distribution within olfactory neurons, particularly in the dendritic knobs where olfactory signaling is initiated .

• Signal transduction coupling: The antibody can be used in concert with G-protein antibodies to investigate coupling specificity. Since OR1K1 functions as a GPCR, understanding its interaction with specific G-proteins (particularly Gαolf in olfactory neurons) is crucial .

• Receptor trafficking studies: Tracking OR1K1 internalization following odorant exposure provides insights into receptor desensitization mechanisms. The antibody can detect changes in surface versus intracellular receptor pools .

• Phosphorylation state analysis: Combined with phospho-specific antibodies, OR1K1 antibody can help determine how receptor phosphorylation affects signaling capacity.

• Heterologous expression systems: As demonstrated in functional studies, OR1K1 antibody can validate receptor expression in systems like HEK293 cells transfected with OR1K1, RTP1S, and Gαolf for signaling studies .

This multi-faceted approach allows detailed characterization of OR1K1's role in olfactory signal transduction .

What methodological approaches optimize OR1K1 detection in challenging experimental contexts?

Detecting OR1K1 in experimental settings presents several challenges that require sophisticated methodological solutions:

• Membrane protein extraction optimization: As a 7-transmembrane protein, OR1K1 requires specialized extraction protocols. Incorporate detergents such as CHAPS, DDM, or Triton X-100 at carefully optimized concentrations to solubilize the receptor while maintaining native conformation .

• Cross-cell type detection strategies: When comparing OR1K1 expression across different cell types (e.g., HEK293, HepG2, HuH7, LNCaP), optimize transfection reagents specifically for each cell line. Research has shown that PEI-MAX works well for HEK293 cells, while Lipofectamine variants are more effective for other cell types .

• Signal amplification techniques: For low abundance detection, implement tyramide signal amplification or high-sensitivity chemiluminescent detection systems. These approaches can significantly improve signal-to-noise ratio in Western blot and immunohistochemistry applications .

• Antibody concentration optimization: Conduct careful titration experiments across a range of concentrations (starting with manufacturer-recommended dilutions of 1:500-2000 for WB) to determine optimal antibody concentration for each experimental system .

• Epitope accessibility enhancement: For fixed tissue or cell preparations, optimize antigen retrieval methods (heat-induced or enzymatic) to maximize epitope exposure, particularly important for membrane-embedded receptors like OR1K1 .

• Co-expression with accessory proteins: In heterologous expression systems, co-transfect with receptor trafficking proteins (RTP1S) to improve surface expression and detection, as demonstrated in functional analysis studies .

These methodological refinements substantially improve OR1K1 detection across diverse experimental contexts .

How can OR1K1 antibody be used to investigate receptor-ligand interactions?

Investigating OR1K1 receptor-ligand interactions requires sophisticated experimental approaches incorporating antibody-based detection methods:

• Post-stimulation conformational analysis: OR1K1 antibody can detect potential conformational changes following odorant binding by comparing epitope accessibility in stimulated versus unstimulated conditions.

• Competitive binding assays: By combining OR1K1 antibody detection with dose-response studies using potential ligands, researchers can identify compounds that induce receptor internalization or conformational changes .

• Proximity ligation assays: This technique can reveal in situ interactions between OR1K1 and downstream signaling components following odorant exposure, providing spatial resolution of signaling events.

• FRET/BRET-based interaction studies: When used with fluorescently tagged G-proteins or β-arrestins, OR1K1 antibody can help validate the components involved in the signaling complex upon ligand binding .

• Calcium imaging correlation: After recording functional calcium responses to potential OR1K1 ligands, cells can be fixed and immunostained with OR1K1 antibody to directly correlate receptor expression with functional responses .

• CRE-luciferase reporter correlation: As shown in functional studies, luciferase reporter assays measuring cAMP production can be combined with OR1K1 antibody detection to link signal transduction to receptor expression levels .

This integrated approach allows researchers to connect OR1K1 expression and localization with functional responses to potential ligands .

What are the technical considerations for using OR1K1 antibody in multi-omics research approaches?

Integrating OR1K1 antibody into multi-omics research strategies requires careful technical considerations:

• Antibody-based proteomics integration: OR1K1 antibody can validate mass spectrometry-based proteomic findings, providing orthogonal confirmation of receptor expression in specific tissues or under varying conditions.

• Transcriptomic-proteomic correlation: Combine RNA-seq data on OR1K1 expression with antibody-based protein detection to investigate post-transcriptional regulation mechanisms affecting receptor abundance .

• Cell-type specific expression profiling: Use OR1K1 antibody in conjunction with cell-type specific markers to create detailed expression maps across olfactory epithelium and potentially ectopic expression sites.

• Sequential extraction protocols: For comprehensive proteomics, develop fractionation protocols that effectively extract membrane proteins like OR1K1 without compromising subsequent detection methods .

• Crosslinking mass spectrometry applications: OR1K1 antibody can help validate crosslinking MS results by confirming the presence of the receptor in isolated protein complexes.

• Single-cell proteomics approaches: Optimize immunofluorescence protocols using OR1K1 antibody for correlation with single-cell transcriptomics, providing multi-level validation of cell-specific expression patterns .

• Post-translational modification analysis: Use OR1K1 antibody to immunoprecipitate the receptor for subsequent analysis of post-translational modifications that may regulate function.

These technical approaches enable comprehensive characterization of OR1K1 biology across multiple analytical platforms .

How can functional analysis data be correlated with OR1K1 antibody detection in heterologous expression systems?

Correlating functional analysis with OR1K1 antibody detection in heterologous expression systems requires sophisticated experimental design:

• Cell line selection optimization: Research has shown differential OR1K1 expression effects across cell lines (HEK293, HepG2, HuH7, LNCaP). Each requires specific transfection reagents and conditions - PEI-MAX (0.1%, pH 7.5) for HEK293, Lipofectamine 2000 for HepG2/HuH7, and Lipofectamine 3000 for LNCaP cells .

• Co-expression system design: For optimal functional studies, co-transfect cells with:

  • FLAG-Rho-tagged OR1K1 in pME18S vector (0.075 μg/well in 96-well format)

  • CRE/luc2PpGL4.29 reporter (0.03 μg/well)

  • RTP1S expression vector (0.03 μg/well)

  • Gαolf expression vector (0.03 μg/well, except in HEK293 cells)

• Luciferase reporter correlation: The Dual-Glo Luciferase Assay System can measure cAMP-dependent signaling, which can be directly correlated with OR1K1 expression levels detected by antibody .

• Surface expression quantification: Use cell surface biotinylation followed by OR1K1 antibody detection to determine what percentage of expressed receptor reaches the plasma membrane .

• Flow cytometry validation: Implement flow cytometry using OR1K1 antibody to quantify expression levels in transfected populations, correlating expression with functional responses.

• Time-course analysis: Combine time-dependent functional measurements with antibody detection to track receptor expression, trafficking, and signaling dynamics.

This methodological approach allows researchers to directly correlate OR1K1 expression levels and localization with functional responses in heterologous systems .