OR1B1 Antibody

What is OR1B1 and why is it significant for research?

OR1B1 (Olfactory Receptor Family 1 Subfamily B Member 1) is a G-protein-coupled receptor encoded by the OR1B1 gene. While primarily classified as an olfactory receptor, research has demonstrated that olfactory receptors are expressed throughout the human body in non-olfactory tissues . As of 2024, OR1B1 remains an orphan receptor, meaning no specific odorants have been identified that bind to it . Its potential functions outside the olfactory system make it an intriguing target for research exploring novel cellular roles of olfactory receptors.

What types of OR1B1 antibodies are currently available for research applications?

Multiple rabbit polyclonal antibodies against OR1B1 are commercially available with validated applications:

How should I optimize Western blot conditions for OR1B1 detection?

For optimal Western blot detection of OR1B1:

• Sample preparation: Use CHAPS buffer (20 mM Tris-HCl, 0.5% CHAPS, pH 8.0) with anti-protease/phosphatase inhibitors for efficient extraction of this transmembrane protein .

• Gel selection: Utilize 4-12% Bis-Tris gradient gels to effectively separate the protein, which has a calculated molecular weight of approximately 36.7 kDa .

• Blocking: Use 5% BSA in Tris-buffered saline with 0.2% Tween 20 (pH 7.4) for 1 hour at room temperature to minimize background .

• Antibody dilution: Begin with a dilution range of 1:500-1:2000 for primary antibody incubation overnight at 4°C .

• Detection system: Chemiluminescent detection systems provide optimal sensitivity for visualizing OR1B1 bands, as demonstrated in studies using the LiCor Western Sure system .

• Controls: Include positive controls from tissues known to express OR1B1, such as A549 or COLO cells, as validated in previous studies .

What are the recommended protocols for immunofluorescence detection of OR1B1?

For immunofluorescence applications:

• Fixation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.2% Triton X-100 for 10 minutes.

• Blocking: Block with 3% BSA in PBS for 1 hour at room temperature.

• Primary antibody: Apply OR1B1 antibody at a dilution of 1:100-1:500 in blocking buffer and incubate overnight at 4°C .

• Secondary antibody: Use fluorophore-conjugated anti-rabbit secondary antibodies (e.g., Alexa Fluor 647) at 1:1000 dilution for 1 hour at room temperature .

• Counterstaining: DAPI (1:5000) for nuclear visualization.

• Imaging parameters: Capture images using confocal microscopy with appropriate filter settings for the secondary antibody fluorophore.

How can I investigate the potential non-olfactory functions of OR1B1 in different tissues?

To study non-olfactory functions of OR1B1:

• Tissue expression profiling: Conduct RT-qPCR analysis across multiple tissues using GAPDH as a reference gene for normalization . Extract RNA using miRNeasy micro isolation kits with DNase treatment to remove genomic DNA contamination.

• Functional characterization: Design calcium imaging experiments using fura-2-AM to detect OR1B1 activation upon stimulation with potential ligands . This methodology has successfully identified functional roles of other ectopically expressed ORs.

• Gene silencing: Implement siRNA strategies targeting OR1B1 followed by functional assays to determine phenotypic consequences in non-olfactory tissues, as demonstrated for other ORs in skin cells .

• Signaling pathway analysis: Use phospho-specific antibodies to detect activation of downstream effectors (particularly cAMP, AKT, and MAPK pathways) following potential OR1B1 activation .

• Tissue-specific knockout models: Generate conditional knockout models to evaluate the physiological relevance of OR1B1 in specific tissues of interest.

What approaches can I use to identify potential ligands for the orphan receptor OR1B1?

For deorphanizing OR1B1:

• High-throughput screening: Establish a stable cell line expressing OR1B1 coupled with a luciferase reporter system driven by a cAMP-responsive element (CRE) to screen libraries of odorant compounds.

• Structure-based virtual screening: Utilize homology modeling based on recently solved OR structures to predict potential ligands that can bind to OR1B1's binding pocket. Molecular dynamics simulations can be employed to assess structural stability of the receptor-ligand complex, as demonstrated for other ORs .

• Metabolomics approach: Analyze metabolites present in tissues with high OR1B1 expression to identify potential endogenous ligands, similar to the identification of fatty acids as ligands for OR51E1 in cardiac tissue .

• Cross-receptor comparison: Examine ligand specificity patterns of phylogenetically related ORs to narrow down potential chemical classes that might activate OR1B1.

• Calcium imaging validation: Confirm potential ligands using calcium imaging in OR1B1-expressing cells, which provides real-time visualization of receptor activation .

How can I address poor surface expression of OR1B1 in heterologous cell systems?

To improve OR1B1 surface expression:

• Co-expression with accessory proteins: Co-transfect cells with RTP1, RTP2, and REEP1, which have been shown to enhance surface trafficking of ORs .

• Consensus sequence engineering: Analyze the OR1B1 sequence for deviations from consensus residues at key positions, particularly focusing on the 66 sites identified as critical for surface expression . Consider introducing mutations to align with consensus sequences, which has dramatically improved surface expression for other ORs.

• Optimization of culture conditions: Incubate transfected cells at 33°C instead of 37°C to facilitate proper protein folding and reduce ER stress.

• Addition of chemical chaperones: Supplement culture medium with 4-phenylbutyric acid (5 mM) or DMSO (1-2%) to stabilize protein folding.

• Fusion tag strategies: Create fusion constructs with rhodopsin N-terminal sequences or Lucy tags, which have been demonstrated to enhance trafficking of difficult-to-express GPCRs .

What are the best methods to validate OR1B1 antibody specificity for research applications?

To ensure antibody specificity:

• Genetic controls: Compare staining patterns between OR1B1-transfected cells and non-transfected controls. Additionally, use CRISPR/Cas9-mediated OR1B1 knockout samples as negative controls.

• Peptide competition assays: Pre-incubate the OR1B1 antibody with the immunizing peptide prior to application to verify signal abolishment.

• Multiple antibody validation: Compare staining patterns using different antibodies raised against distinct epitopes of OR1B1.

• Correlation with mRNA expression: Perform parallel RT-qPCR to correlate protein detection with mRNA expression levels across different tissues and cell types.

• Cross-reactivity assessment: Test antibody reactivity against closely related olfactory receptors, particularly those within the OR1 subfamily, to confirm specificity.

How can I investigate the structural determinants affecting OR1B1 stability and surface expression?

To study OR1B1 structural determinants:

• Homology modeling: Generate OR1B1 structural models based on recently solved olfactory receptor structures , focusing on conserved residues at positions 4.53 and 5.47 which have been identified as critical for stability of other ORs .

• Molecular dynamics simulations: Perform MD simulations of wild-type and mutant OR1B1 models in explicit lipid bilayers to assess structural stability, with particular attention to RMSD variations in the transmembrane and extracellular domains .

• Site-directed mutagenesis: Create a panel of OR1B1 mutants targeting conserved residues and assess their impact on surface expression and function through flow cytometry and functional assays.

• Thermal stability assays: Implement nanoDSF (differential scanning fluorimetry) to measure thermal stability of purified OR1B1 variants.

• Crosslinking mass spectrometry: Apply this technique to capture intramolecular interactions critical for OR1B1 stability and folding.

What experimental approaches can determine if OR1B1 has physiological functions in non-olfactory tissues?

To investigate non-olfactory functions:

• Tissue-specific gene expression analysis: Perform RT-qPCR and RNAscope in situ hybridization to quantify and localize OR1B1 expression across multiple tissues, as demonstrated for other ectopically expressed ORs .

• Functional assays: Based on the tissue of interest, implement relevant functional assays:

  • For potential roles in blood cells: Analyze chemotaxis, proliferation, and apoptosis

  • For skin cells: Assess proliferation, migration, and differentiation

  • For other tissues: Evaluate tissue-specific functions (e.g., contractility in muscle cells) upon OR1B1 activation or inhibition

• Signaling pathway characterization: Analyze activation of downstream effectors using phospho-kinase arrays and Western blotting to map signaling cascades initiated by OR1B1 .

• Physiological measurements: In ex vivo tissue preparations expressing OR1B1, test the effects of potential ligands on tissue-specific physiological parameters, similar to studies of OR51E1 in cardiac tissue .

• In vivo models: Generate conditional OR1B1 knockout models to assess physiological consequences in specific tissues of interest.