OR13C4 Antibody

What is OR13C4 and why is it studied in research?

OR13C4 (Olfactory Receptor Family 13 Subfamily C Member 4) is a G protein-coupled receptor primarily expressed in olfactory sensory neurons. This receptor belongs to the largest gene family in the human genome, responsible for odor detection and signal transduction. Research on OR13C4 contributes to understanding olfactory system development, signal transduction mechanisms, and potential extranasal functions of olfactory receptors. The interest in OR13C4 has expanded as studies have revealed ectopic expression of olfactory receptors in non-olfactory tissues, suggesting roles beyond conventional odor detection. Antibodies against OR13C4 enable researchers to detect and quantify this protein across different experimental contexts, making them essential tools for advancing our knowledge of sensory biology and related fields .

What are the key characteristics of commercially available OR13C4 antibodies?

The primary OR13C4 antibodies available for research are polyclonal antibodies produced in rabbits. These antibodies target epitopes within the amino acid range 201-250 of the human OR13C4 protein, which has a molecular weight of approximately 35 kDa. They are typically provided as affinity isolated antibodies in buffered aqueous solutions, with concentrations around 1 mg/mL. These antibodies demonstrate reactivity to human OR13C4 protein, with some products also showing cross-reactivity with monkey orthologs. The antibodies are available in unconjugated forms and are designed for applications including Western blot, immunofluorescence, and ELISA. The polyclonal nature of these antibodies provides robust signal detection across multiple epitopes, though this can sometimes lead to higher background compared to monoclonal alternatives .

What are the optimal dilution ratios for different OR13C4 antibody applications?

The optimal dilution ratios for OR13C4 antibodies vary significantly depending on the experimental application, specific antibody product, and sample characteristics. For Western blot applications, dilutions ranging from 1:500 to 1:2000 are typically recommended, with most laboratories starting at 1:1000 and adjusting based on signal-to-noise ratios. For immunofluorescence applications, a more concentrated solution is typically required, with recommended dilutions of 1:100 to 1:500. ELISA applications generally require more dilute antibody preparations, with recommended dilutions around 1:5000. These ratios should be considered starting points rather than absolute values, as optimal concentrations may need to be determined empirically for each experimental system. Factors such as antigen abundance, tissue type, fixation method, and detection system sensitivity all influence the optimal antibody concentration. A titration experiment across a range of dilutions is strongly recommended when establishing a new protocol or working with a new lot of antibody .

How should researchers optimize protein extraction protocols for OR13C4 detection in Western blotting?

Optimizing protein extraction for OR13C4 detection requires special consideration due to its nature as a transmembrane G protein-coupled receptor. The protocol should incorporate several key elements: (1) Use of specialized lysis buffers containing 1-2% non-ionic detergents (such as Triton X-100, NP-40, or CHAPS) to effectively solubilize membrane proteins; (2) Inclusion of protease inhibitor cocktails to prevent degradation, particularly important for potentially labile receptors like OR13C4; (3) Gentle homogenization methods to maintain protein integrity while ensuring effective extraction from membrane fractions; (4) Avoidance of excessive heating during sample preparation, as transmembrane proteins can aggregate at high temperatures; (5) Preparation of fresh samples whenever possible, as freeze-thaw cycles can degrade membrane proteins. When running the gel, researchers should consider using gradient gels (4-12% or 4-20%) to improve separation of the 35 kDa OR13C4 protein from other similarly sized proteins. Following transfer, membrane blocking should be performed with either 5% non-fat dry milk or 3-5% BSA in TBS-T, with the latter potentially providing better results for phosphorylation-specific detection .

What are the recommended immunofluorescence protocols for OR13C4 localization studies?

For optimal OR13C4 visualization using immunofluorescence, researchers should follow a protocol tailored to transmembrane protein detection. Begin with fixation using 4% paraformaldehyde for 15-20 minutes at room temperature, which preserves cellular architecture while maintaining epitope accessibility. A critical step is the permeabilization process: use 0.1-0.3% Triton X-100 for 10 minutes to allow antibody access to intracellular domains while preventing over-permeabilization that could disrupt membrane integrity. For blocking, use 5-10% normal serum from the same species as the secondary antibody in PBS with 0.1% Tween-20 for 1 hour at room temperature. Apply the primary OR13C4 antibody at dilutions between 1:100 and 1:500 in blocking buffer, incubating overnight at 4°C to maximize specific binding. For detection, use fluorophore-conjugated secondary antibodies at 1:500-1:1000, incubating for 1-2 hours at room temperature in the dark. Include DAPI (1:1000) for nuclear counterstaining. Between each step, perform three 5-minute washes with PBS containing 0.1% Tween-20. When imaging, use confocal microscopy with appropriate filter sets to accurately visualize membrane localization patterns, which is particularly important for resolving the subcellular distribution of this transmembrane receptor .

How can researchers validate the specificity of OR13C4 antibodies?

Validating OR13C4 antibody specificity requires a multi-faceted approach due to the high sequence homology among olfactory receptors. First, researchers should implement a peptide competition assay where the antibody is pre-incubated with the immunizing peptide (corresponding to amino acids 201-250 of OR13C4) before application to the sample; specific binding should be significantly reduced. Second, utilize positive and negative control tissues or cell lines with known OR13C4 expression levels, confirmed by orthogonal methods such as qPCR. Third, employ knockout or knockdown validation where OR13C4 expression is eliminated via CRISPR-Cas9 or siRNA technologies; the antibody signal should be accordingly reduced or eliminated. Fourth, consider Western blot analysis to confirm that the detected protein matches the expected molecular weight of 35 kDa, though post-translational modifications may alter this. Finally, cross-validation using multiple antibodies targeting different epitopes within OR13C4 can provide additional confidence in specificity. Researchers should be particularly vigilant about potential cross-reactivity with similar olfactory receptors such as OR2K1, OR37F, and OR9-7, which have been noted as alternative names in some database entries, suggesting potential sequence or structural similarities .

What controls should be included when performing OR13C4 immunodetection experiments?

A robust experimental design for OR13C4 immunodetection requires multiple controls to ensure reliability and reproducibility. For primary antibody validation, include: (1) A no-primary antibody control to assess background from non-specific secondary antibody binding; (2) An isotype control using non-specific rabbit IgG at the same concentration as the OR13C4 antibody to evaluate background from non-specific binding of rabbit immunoglobulins; (3) A peptide competition control as described earlier. For sample validation, include: (4) Positive control tissues/cells with confirmed OR13C4 expression (olfactory epithelium is ideal, though certain non-olfactory tissues may also express this receptor); (5) Negative control tissues/cells lacking OR13C4 expression; (6) If possible, OR13C4 overexpression systems to confirm antibody sensitivity. For technical validation: (7) Include loading controls appropriate to the experimental context (β-actin, GAPDH for total protein; Na⁺/K⁺-ATPase or calnexin for membrane fractions); (8) For quantitative analyses, prepare a standard curve using recombinant OR13C4 protein if available. These controls help distinguish true OR13C4 signal from technical artifacts, non-specific binding, or cross-reactivity with related olfactory receptors .

How do storage conditions affect OR13C4 antibody performance over time?

The storage conditions of OR13C4 antibodies significantly impact their long-term performance and reliability. These antibodies should be stored at -20°C for long-term preservation, with aliquoting strongly recommended to avoid repeated freeze-thaw cycles that can cause antibody degradation through protein denaturation and aggregation. Once thawed for use, working aliquots can be stored at 4°C for up to two weeks, though sensitivity may gradually decrease during this period. The antibody formulation typically includes 50% glycerol, which prevents freezing solid at -20°C and allows for easier handling without complete thawing. The presence of 0.02% sodium azide in the formulation helps prevent microbial contamination during storage but should be noted as potentially incompatible with certain enzyme-based detection systems. BSA (0.5%) in the formulation helps stabilize the antibody proteins during storage and freeze-thaw transitions. Researchers should monitor for signs of antibody deterioration, including visible precipitates, unusual cloudiness, or unexpected shifts in experimental results compared to previous lots. Maintaining proper storage records, including freeze-thaw cycles and lot numbers, can help trace performance changes to storage conditions. When long-term studies are planned, creating sufficient single-use aliquots from the same lot is advisable to ensure consistency throughout the research project .

How can researchers address weak or absent signal in OR13C4 Western blots?

When encountering weak or absent signals in OR13C4 Western blots, researchers should systematically evaluate and optimize several parameters. First, address protein extraction efficiency by using stronger lysis buffers (consider RIPA or specialized membrane protein extraction buffers) and longer extraction times, as OR13C4's transmembrane nature can make it difficult to solubilize. Second, evaluate protein loading and transfer by using reversible total protein stains on membranes to confirm sufficient protein transfer. Third, modify antibody incubation conditions by increasing primary antibody concentration (try 1:250 instead of 1:1000), extending incubation time to overnight at 4°C, or adding 0.05% SDS to the antibody dilution buffer to enhance epitope accessibility. Fourth, optimize detection sensitivity by using enhanced chemiluminescence (ECL) substrates with higher sensitivity or switching to fluorescent secondary antibodies with direct digital imaging. Fifth, consider membrane type and blocking agents, as PVDF membranes may provide better protein retention than nitrocellulose for some applications, and BSA blocking might be preferable to milk for certain epitopes. Finally, evaluate sample preparation techniques, including avoiding excessive heating during sample preparation (95°C for 5 minutes may be sufficient) and using fresh DTT or β-mercaptoethanol in loading buffer to ensure proper protein denaturation and epitope exposure .

What strategies can resolve high background in OR13C4 immunofluorescence staining?

High background in OR13C4 immunofluorescence staining can significantly compromise data interpretation and requires systematic troubleshooting. First, optimize blocking conditions by extending blocking time to 2 hours, increasing blocking agent concentration to 5-10%, or switching between different blocking agents (normal serum, BSA, casein) to identify optimal conditions for your specific tissue. Second, modify antibody incubation parameters by diluting the primary antibody further (try 1:300-1:500 instead of 1:100), adding 0.1-0.3% Triton X-100 to antibody dilution buffers to reduce non-specific binding, and extending wash steps to 15 minutes with 3-5 changes of wash buffer. Third, evaluate fixation protocols, as excessive fixation can increase autofluorescence; consider optimizing paraformaldehyde concentration (2-4%) and fixation time (10-20 minutes). Fourth, implement additional background reduction techniques such as including 0.1-0.3% Tween-20 in all buffers, pre-absorbing primary antibodies with tissue homogenates from negative control samples, or treating sections with 0.1-1% sodium borohydride before blocking to reduce autofluorescence. Fifth, consider tissue-specific issues by using Sudan Black B (0.1-0.3%) to reduce lipofuscin autofluorescence in certain tissues or implementing automated background subtraction during image acquisition with appropriate controls. Finally, always include a secondary-only control to distinguish between primary antibody-specific background and issues with the secondary antibody or autofluorescence .

How should researchers interpret multiple bands in OR13C4 Western blots?

Multiple bands in OR13C4 Western blots require careful analysis to distinguish between genuine biological signals and technical artifacts. First, evaluate the molecular weights of observed bands: the primary OR13C4 band should appear at approximately 35 kDa. Bands at ~70 kDa might represent dimerized receptor, while bands at ~25-28 kDa could indicate proteolytic fragments. Second, consider post-translational modifications: glycosylation can add 5-15 kDa to the apparent molecular weight, phosphorylation may cause slight shifts, and ubiquitination can create ladder-like patterns at higher molecular weights. Third, perform validation experiments: peptide competition assays should reduce all specific bands, deglycosylation with PNGase F can collapse multiple glycoform bands, and membrane protein enrichment should enhance signals from genuine receptor bands. Fourth, analyze sample preparation effects: insufficient denaturation (especially common with membrane proteins) can cause anomalous migration, while excessive heating can cause aggregation appearing as high-molecular-weight bands. Fifth, consider cross-reactivity possibilities: due to the high homology among olfactory receptors, bands may represent related family members; this can be investigated using tissues with known differential expression of OR family members. Finally, evaluate technical parameters: overexposed blots can reveal minor cross-reactive bands, while low-quality transfer can cause smearing or multiple bands. Careful optimization of SDS-PAGE parameters (including gel percentage, running time, and voltage) may improve band resolution and aid interpretation .

How can OR13C4 antibodies be utilized in co-immunoprecipitation studies of receptor-protein interactions?

Co-immunoprecipitation (Co-IP) studies with OR13C4 antibodies require specialized approaches to accommodate the challenges of working with membrane-bound G protein-coupled receptors. For successful Co-IP experiments, researchers should use mild lysis conditions that preserve protein-protein interactions while effectively solubilizing membrane proteins; buffers containing 0.3-1% digitonin, 0.5-1% NP-40, or 0.5-1% CHAPS are often effective. The lysis buffer should include physiological salt concentrations (150 mM NaCl) and pH (7.4), with protease and phosphatase inhibitors to preserve native interactions. Pre-clearing lysates with protein A/G beads reduces non-specific binding. When coupling the OR13C4 antibody to beads, covalent cross-linking with dimethyl pimelimidate may prevent antibody leaching and reduce background. Incubation conditions should be optimized (typically 2-4 hours at 4°C) to balance complete capture with minimal disruption of protein complexes. For elution, consider using the immunizing peptide for specific displacement rather than harsh denaturing conditions that might disrupt protein complexes. When analyzing results, focus on known GPCR-associated proteins such as G proteins (Gαolf), arrestins, and receptor kinases as positive controls. To confirm specificity, perform reverse Co-IP experiments using antibodies against suspected interaction partners to pull down OR13C4. This approach can reveal novel protein interactions involved in olfactory signal transduction and potentially identify unexpected signaling partners in non-olfactory tissues .

What considerations are important when designing quantitative analyses of OR13C4 expression levels?

Quantitative analysis of OR13C4 expression requires careful consideration of several technical and biological factors to ensure reliable results. First, establish a validated detection system: for Western blot quantification, create a standard curve using recombinant OR13C4 protein (if available) to ensure measurements fall within the linear range of detection; for immunofluorescence quantification, use consistent image acquisition parameters and analyze images with appropriate software that can distinguish membrane from cytoplasmic staining. Second, implement rigorous normalization strategies: normalize Western blot data to multiple housekeeping proteins or total protein stains (e.g., REVERT or Ponceau S) rather than single reference proteins; for immunofluorescence, normalize to membrane markers appropriate for the subcellular localization of OR13C4. Third, account for OR13C4's cell-type specificity by using markers to identify specific cell populations when analyzing heterogeneous tissues. Fourth, be aware of potential confounding factors: circadian rhythms may affect olfactory receptor expression, so standardize tissue collection times; olfactory receptors can be regulated by their ligands, so control for potential odorant exposure. Fifth, employ complementary techniques such as qRT-PCR to correlate protein levels with mRNA expression, though remembering that these may not always directly correlate due to post-transcriptional regulation. Finally, use statistical approaches appropriate for the potentially high variability in membrane protein expression, including sufficient biological replicates (minimum n=3, ideally n≥5) and appropriate statistical tests for the data distribution patterns observed .

How can researchers apply OR13C4 antibodies in studying receptor trafficking and internalization?

Investigating OR13C4 trafficking and internalization requires specialized experimental approaches leveraging the capabilities of OR13C4 antibodies. First, implement pulse-chase immunofluorescence studies using primary OR13C4 antibodies to label surface receptors in live cells at 4°C (to prevent internalization), followed by warming to 37°C to permit trafficking, then fixing at different time points to track receptor movement. Second, establish dual-labeling protocols using OR13C4 antibodies in conjunction with markers for different cellular compartments: early endosomes (EEA1), recycling endosomes (Rab11), lysosomes (LAMP1), and the trans-Golgi network (TGN46) to map trafficking pathways. Third, develop quantitative internalization assays using biotinylated OR13C4 antibodies to label surface receptors, followed by acid wash to remove remaining surface antibodies, and quantification of internalized biotinylated antibodies via ELISA or Western blot. Fourth, consider live-cell imaging approaches by carefully conjugating OR13C4 antibodies with pH-sensitive fluorophores like pHrodo that increase fluorescence in acidic environments, allowing real-time visualization of receptor internalization into endosomal compartments. Fifth, implement super-resolution microscopy techniques like STORM or PALM using appropriately labeled secondary antibodies to resolve the nanoscale organization of OR13C4 in membrane microdomains and trafficking vesicles. When designing these experiments, researchers should be aware of potential antibody-induced receptor clustering or activation, which might influence trafficking patterns, and include appropriate controls such as isotype-matched non-specific antibodies to distinguish between natural and induced receptor behaviors .

How might newer antibody engineering technologies enhance OR13C4 research capabilities?

Advanced antibody engineering technologies offer promising opportunities to overcome current limitations in OR13C4 research. First, single-domain antibodies (nanobodies) derived from camelid antibodies could provide superior access to conformational epitopes on this transmembrane receptor due to their small size (approximately 15 kDa), potentially revealing functionally relevant structural states. Second, site-specific conjugation technologies using unnatural amino acids or enzymatic modifications can generate homogeneous antibody conjugates with precisely positioned fluorophores, offering improved resolution in advanced microscopy applications. Third, switchable antibody systems based on chemical regulation could enable temporal control of OR13C4 detection, allowing pulse-chase experiments with greater precision than traditional approaches. Fourth, bispecific antibodies targeting both OR13C4 and key signaling partners could enable direct visualization of protein-protein interactions in situ. Fifth, phage display technologies could generate highly specific monoclonal antibodies against difficult-to-access epitopes of OR13C4, improving specificity beyond current polyclonal offerings. While these approaches hold significant promise, researchers should carefully validate any newer antibody technologies against established gold standards. The potential benefits of these advanced tools include improved spatial resolution in localization studies, better discrimination between closely related olfactory receptors, and enhanced capabilities for studying the dynamic processes of receptor activation and trafficking .

What role might OR13C4 antibodies play in understanding olfactory receptor expression in non-olfactory tissues?

OR13C4 antibodies are becoming invaluable tools for exploring the emerging field of ectopic olfactory receptor expression in non-olfactory tissues. Accumulating evidence suggests olfactory receptors, including OR13C4, may be expressed outside the nasal epithelium and serve non-chemosensory functions. Researchers can leverage OR13C4 antibodies to perform systematic tissue screening via tissue microarrays to identify unexpected expression patterns across diverse human tissues. Once identified, detailed characterization of cell-type specific expression within these tissues can be achieved through co-localization studies with cell-type specific markers using multiplexed immunofluorescence or immunohistochemistry. OR13C4 antibodies can further help elucidate the subcellular localization of the receptor in these non-olfactory contexts, which may differ from the canonical patterns observed in olfactory neurons. Beyond detection, these antibodies enable functional investigations through receptor neutralization experiments, potentially blocking novel signaling pathways. Additionally, OR13C4 antibodies can facilitate the identification of tissue-specific ligands through co-immunoprecipitation of the receptor with bound molecules. In disease contexts, these antibodies may reveal altered expression patterns that could serve as biomarkers or therapeutic targets. The systematic application of OR13C4 antibodies across diverse tissues represents a promising approach to uncovering novel functions of this receptor beyond its conventional role in olfaction .

How can researchers integrate OR13C4 antibody-based detection with emerging single-cell technologies?

Integrating OR13C4 antibody-based detection with cutting-edge single-cell technologies opens new frontiers for understanding receptor heterogeneity and function at unprecedented resolution. First, researchers can adapt OR13C4 antibodies for use in cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) by conjugating them with oligonucleotide barcodes, enabling simultaneous protein detection and transcriptome analysis at the single-cell level. This approach can reveal correlations between OR13C4 protein expression and broader transcriptional programs. Second, imaging mass cytometry using metal-conjugated OR13C4 antibodies allows multiplexed protein detection (30+ targets) while preserving spatial information, enabling complex phenotyping of OR13C4-expressing cells within their tissue microenvironment. Third, integrating OR13C4 immunofluorescence with laser capture microdissection enables selective isolation of OR13C4-positive cells for downstream molecular analyses, including proteomics or transcriptomics. Fourth, in situ sequencing approaches combined with OR13C4 immunodetection can map the spatial relationship between receptor expression and relevant mRNAs. Fifth, microfluidic systems can isolate single OR13C4-positive cells (identified by antibody labeling) for functional studies, including calcium imaging or electrophysiology, directly linking receptor expression to functional properties. These integrated approaches can address fundamental questions about the heterogeneity of OR13C4 expression, its co-expression with signaling partners, and cell-to-cell variability in receptor distribution and function, potentially revealing previously unrecognized cellular subtypes and functional states .