OR5K2 Antibody

What is OR5K2 and what is its physiological function?

OR5K2 (also known as Olfactory receptor OR3-9) is a member of the olfactory receptor family that interacts with odorant molecules in the nose to initiate neuronal responses triggering smell perception. As a G-protein-coupled receptor (GPCR), OR5K2 features a characteristic 7-transmembrane domain structure shared with many neurotransmitter and hormone receptors. This protein is responsible for recognizing specific odorants and mediating G protein-coupled signal transduction, ultimately resulting in the perception of particular smells . The receptor is encoded by a single coding-exon gene and forms part of the largest gene family in the human genome with a cellular localization in the cell membrane as a multi-pass membrane protein .

How should I select an appropriate OR5K2 antibody for my specific research application?

When selecting an OR5K2 antibody, consider multiple factors: (1) target application compatibility (different antibodies are optimized for specific techniques like Western blot, ELISA, or immunohistochemistry); (2) epitope location (available antibodies target different regions, such as amino acids 6-24 or 30-110); (3) species reactivity (some antibodies recognize only human OR5K2 while others cross-react with mouse and rat homologs); and (4) conjugation requirements (antibodies are available with various conjugates like HRP, FITC, or biotin for different detection methods) . For example, the antibody from antibodies-online (ABIN7382922) targets amino acids 6-24 and is optimized for ELISA applications, while St John's Laboratory antibody (STJ192754) targets amino acids 30-110 and is suitable for both Western blotting and ELISA with human, rat, and mouse reactivity .

What experimental controls are essential when working with OR5K2 antibodies?

Essential controls for OR5K2 antibody experiments include: (1) positive tissue controls (olfactory epithelium where OR5K2 is naturally expressed); (2) negative tissue controls (tissues not expressing OR5K2); (3) peptide competition assays (pre-incubating the antibody with immunizing peptide should abolish specific signal); (4) isotype controls (using non-specific IgG from the same host species); and (5) secondary-only controls (omitting primary antibody) . For advanced validation, consider using OR5K2 overexpression systems compared to non-transfected cells, or employing siRNA knockdown of OR5K2 to confirm antibody specificity. These controls help distinguish true OR5K2 signal from background or cross-reactivity with related olfactory receptors, which is particularly important given the high sequence similarity within this large gene family .

How should I optimize Western blot protocols for detecting OR5K2?

Optimizing Western blot protocols for OR5K2 detection requires special consideration of its membrane protein nature. Begin with efficient membrane protein extraction using appropriate detergent-based lysis buffers containing protease inhibitors. For sample preparation, avoid boiling which can cause membrane protein aggregation; instead incubate at 37-50°C for 10-30 minutes. Use 10-12% polyacrylamide gels for optimal separation of the ~36 kDa OR5K2 protein. For antibody incubation, start with the manufacturer's recommended dilution (typically 1:1000-1:5000 for Western blot applications) . Incorporate extended blocking (5% non-fat milk or BSA for 1-2 hours) and washing steps to minimize background. Consider using high-sensitivity detection systems, as endogenous OR5K2 expression may be relatively low in non-olfactory tissues. If weak signals persist, membrane staining with Ponceau S before blocking can confirm successful protein transfer .

What are the optimal conditions for immunohistochemical detection of OR5K2?

For optimal immunohistochemical detection of OR5K2, begin with appropriate fixation—4% paraformaldehyde for 24-48 hours is recommended for tissue samples. For formalin-fixed paraffin-embedded tissues, heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is essential to expose OR5K2 epitopes masked during fixation. Since OR5K2 is a membrane protein, permeabilization with 0.1-0.3% Triton X-100 improves antibody accessibility. Block with 5-10% normal serum from the secondary antibody species for at least 1 hour to reduce non-specific binding. Incubate with primary OR5K2 antibody at 4°C overnight (starting with dilutions recommended for IHC, typically 1:100-1:500), followed by appropriate HRP-conjugated or fluorescently-labeled secondary antibody . Include sudan black treatment if using fluorescent detection to reduce autofluorescence, particularly important in olfactory tissues that can exhibit high background signal .

How can I quantify OR5K2 expression levels in different experimental conditions?

Quantification of OR5K2 expression can be performed through multiple approaches depending on experimental needs. For protein-level quantification, densitometric analysis of Western blots is suitable, normalizing OR5K2 band intensity to housekeeping proteins like β-actin or GAPDH using image analysis software. For higher sensitivity and throughput, consider sandwich ELISA assays using OR5K2 antibodies with optimal dilutions (1:5000-1:20000) . Flow cytometry can assess OR5K2 surface expression in cell populations when using antibodies targeting extracellular domains. For spatial information, quantitative immunofluorescence microscopy with appropriate image analysis software can measure intensity per cell or region. For transcript-level quantification, complement protein data with qRT-PCR using validated OR5K2-specific primers. This multi-modal approach to quantification provides more robust evidence of expression changes across experimental conditions while controlling for potential antibody limitations .

How can I investigate OR5K2 interaction with G proteins using antibody-based techniques?

Investigating OR5K2 interactions with G proteins requires specialized antibody-based approaches. Co-immunoprecipitation (Co-IP) represents the primary method, using OR5K2 antibody to pull down the receptor complex followed by Western blotting with antibodies against suspected G protein partners (typically Gαolf in olfactory signaling). This requires antibodies suitable for immunoprecipitation, often with higher affinity than those used for simple detection. Proximity ligation assays (PLA) offer an alternative approach, using OR5K2 antibodies together with G protein antibodies to generate fluorescent signals only when proteins are within 40nm of each other, enabling visualization of interactions in situ. For dynamic interaction studies, consider FRET/BRET approaches using tagged OR5K2 constructs. When designing these experiments, careful consideration of epitope location is crucial—antibodies targeting intracellular loops or C-terminal domains of OR5K2 are typically more suitable for studying G protein interactions, as these regions mediate G protein coupling .

What strategies can address potential cross-reactivity with other olfactory receptors?

Addressing cross-reactivity with other olfactory receptors requires multi-faceted validation strategies. First, perform in silico analysis to identify olfactory receptors with high sequence homology to OR5K2, particularly OR5K1 which is an important paralog . Use these insights to design competitive peptide blocking experiments with peptides from potential cross-reactive receptors. Employ orthogonal detection methods by comparing antibody-based detection with transcript-specific methods like RNA-Seq or qPCR with highly specific primers. Consider Western blot analysis under high-stringency conditions (higher detergent concentration in wash buffers) to reduce low-affinity binding to homologous receptors. For definitive specificity assessment, use CRISPR/Cas9-mediated OR5K2 knockout models as negative controls, or heterologous expression systems with individually expressed olfactory receptors. When cross-reactivity cannot be eliminated, clearly acknowledge these limitations in experimental interpretations and consider using multiple antibodies targeting different epitopes to increase confidence in results .

How can post-translational modifications of OR5K2 be investigated using antibody-based approaches?

Investigating post-translational modifications (PTMs) of OR5K2 requires specialized antibodies and detection strategies. For phosphorylation analysis, use phospho-specific antibodies targeting common phosphorylation sites in GPCRs (particularly serine/threonine residues in the C-terminal domain) or generic phospho-serine/threonine antibodies after OR5K2 immunoprecipitation. For glycosylation assessment, compare OR5K2 molecular weight before and after treatment with glycosidases like PNGase F or EndoH, detecting mobility shifts via Western blot. Mass spectrometry analysis of immunoprecipitated OR5K2 provides the most comprehensive PTM mapping. For ubiquitination studies, co-immunoprecipitation with OR5K2 antibodies followed by ubiquitin detection, or vice versa, can reveal modification patterns. When investigating PTM dynamics, consider using cell treatments that affect specific modifications (phosphatase inhibitors, glycosylation inhibitors, or proteasome inhibitors) followed by OR5K2 immunoblotting to observe resulting changes in banding patterns .

What are the key differences between commercially available OR5K2 antibodies?

Commercial OR5K2 antibodies differ significantly in several important aspects that can impact experimental outcomes. The primary differences include: (1) Epitope targets: antibodies from antibodies-online target amino acids 6-24, while St John's Laboratory antibodies target residues 30-110, potentially affecting detection of specific protein conformations or splice variants; (2) Host organisms: while most available antibodies are raised in rabbits, their polyclonal nature means each lot may have unique characteristics; (3) Validated applications: antibodies from CUSABIO are optimized for Western blot with specific recommended dilutions (1:1000-1:5000), Abcepta's antibody is validated for immunohistochemistry and immunofluorescence, and others have ELISA-specific validations; (4) Species reactivity: some antibodies react only with human OR5K2 while others (like St John's Laboratory antibody) recognize human, rat and mouse homologs; and (5) Conjugation options: antibodies are available unconjugated or with various conjugates (HRP, FITC, biotin) for specialized applications .

What limitations should researchers be aware of when interpreting OR5K2 antibody results?

Researchers should be cognizant of several limitations when interpreting OR5K2 antibody results. First, the high sequence homology with other olfactory receptors creates potential for cross-reactivity, particularly with the paralog OR5K1 . Second, as polyclonal antibodies, batch-to-batch variability may influence detection sensitivity and background levels, necessitating validation of each lot. Third, as a GPCR, OR5K2 undergoes conformational changes during activation that may alter epitope accessibility, potentially resulting in differential detection of active versus inactive receptor states. Fourth, endogenous expression of OR5K2 may be very low in non-olfactory tissues, leading to detection challenges that could be misinterpreted as absence of expression. Finally, standard fixation and permeabilization protocols may not optimally preserve OR5K2's native conformation, and inconsistent protocols across laboratories contribute to reproducibility challenges . These limitations underscore the importance of comprehensive controls and orthogonal validation approaches when studying OR5K2.

What are the recommended storage and handling conditions for OR5K2 antibodies?

Most commercial OR5K2 antibodies require specific storage and handling conditions to maintain optimal activity. Store antibodies at -20°C for long-term preservation, avoiding repeated freeze-thaw cycles that can degrade antibody performance. Many OR5K2 antibodies are supplied in stabilizing solutions containing 50% glycerol and small amounts (0.02-0.09%) of sodium azide as preservative . When handling, always use clean pipettes dedicated to antibody work to prevent contamination. For working aliquots, store at 4°C for up to two weeks when reconstituted in sterile PBS or appropriate diluent . Avoid exposing antibodies to direct light, particularly those with fluorescent conjugates. When preparing dilutions, use high-quality, sterile buffers with appropriate blocking agents (BSA, normal serum) to minimize non-specific binding. Document lot numbers, receipt dates, and freezing/thawing cycles to track antibody performance over time .

What is the molecular and structural basis for OR5K2 antibody recognition?

The molecular basis for OR5K2 antibody recognition centers on the protein's seven-transmembrane domain structure typical of G-protein-coupled receptors. Commercial antibodies target different epitopes, with some focusing on amino acids 6-24 (N-terminal region) and others on residues 30-110 , which likely include portions of the first extracellular loop and transmembrane domains. The antibody recognition depends on the three-dimensional presentation of these epitopes, which may be influenced by the membrane environment for this integral membrane protein. The calculated molecular weight of OR5K2 is approximately 35,763 Da , providing a reference point for antibody validation via Western blot. The protein's cellular localization in the cell membrane as a multi-pass membrane protein means that certain epitopes may be more accessible than others depending on their orientation (extracellular, intracellular, or within the membrane). This structural arrangement necessitates careful consideration when selecting antibodies for specific applications, particularly for techniques requiring native protein conformations .

What is known about OR5K2 expression patterns across different tissues and species?

OR5K2 expression exhibits characteristic tissue and species-specific patterns relevant to antibody-based detection strategies. As an olfactory receptor, OR5K2 shows highest expression in olfactory epithelium within the nasal cavity, making this tissue an ideal positive control for antibody validation. The olfactory receptor gene family nomenclature is species-independent , complicating cross-species comparisons, though some antibodies show cross-reactivity with rat and mouse homologs . While primarily expressed in olfactory tissues, emerging evidence suggests potential ectopic expression in selected non-olfactory tissues at lower levels, though this requires rigorous validation with highly specific antibodies. The gene has been assigned Gene ID 402135 and UniProt ID Q8NHB8 , with OR3-9 as an alternative designation . OR5K2 belongs to the OR5 subfamily, with OR5K1 representing an important paralog that may share significant sequence homology , highlighting the need for highly specific antibody reagents when distinguishing between closely related family members in expression studies.

Table 1: Commercial OR5K2 Antibody Comparison

Table 2: Recommended Application-Specific Dilutions

Table 3: OR5K2 Protein Characteristics