OR4C15 Antibody

Basic Research Questions

• What is OR4C15 and why is it studied in olfactory research?

  OR4C15 (Olfactory Receptor Family 4 Subfamily C Member 15) is a G-protein-coupled receptor (GPCR) encoded by the OR4C15 gene located on chromosome 11q11 . It functions as an odorant receptor that interacts with odorant molecules in the nose to initiate neuronal responses that trigger smell perception .

  The protein is part of the largest gene family in the genome and shares a 7-transmembrane domain structure with many neurotransmitter and hormone receptors . OR4C15 is primarily studied in olfactory research to understand the molecular mechanisms of smell perception, receptor-ligand interactions, and signal transduction pathways in the olfactory system.

• What are the key specifications of commercially available OR4C15 antibodies?

  Most commercially available OR4C15 antibodies share these specifications:

  Characteristic	Specification
  Host	Rabbit (most common)
  Clonality	Polyclonal (predominant)
  Reactivity	Human (primary), with some cross-reactivity to Rat and Mouse
  Applications	WB, IF/ICC, ELISA
  Recommended dilutions	WB: 1/500-1/3000, IF/ICC: 1/100-1/1000, ELISA: 1/40000
  Immunogen	Typically synthetic peptide from C-terminal region of human OR4C15 (often AA range 261-310)
  Molecular weight	~34-35 kDa
  Form	Liquid in PBS with glycerol (usually 50%), and sodium azide (0.02%)
  Storage	-20°C, avoid repeated freeze/thaw cycles

• What are the most common applications for OR4C15 antibodies in research?

  OR4C15 antibodies are primarily used in three main applications :

  • Western Blot (WB): For detecting OR4C15 protein in cell or tissue lysates, typically at dilutions of 1:500-1:3000. Western blot analysis has been documented with extracts from cell lines such as HepG2 and Jurkat cells .

  • Immunofluorescence (IF)/Immunocytochemistry (ICC): For localizing OR4C15 protein in fixed cells or tissue sections, typically used at dilutions of 1:100-1:1000. This application is valuable for studying the subcellular localization of OR4C15, which is primarily a cell membrane multi-pass protein .

  • ELISA: For quantitative detection of OR4C15 protein in various samples, typically used at higher dilutions around 1:40000 .

  Less common but documented applications include immunohistochemistry (IHC) with paraffin-embedded sections .

Intermediate Research Questions

• How should I optimize Western blot protocols for OR4C15 detection?

  Optimizing Western blot protocols for OR4C15 detection requires attention to several key parameters:

  1. Sample preparation: For membrane proteins like OR4C15, use lysis buffers containing non-ionic detergents (0.5-1% Triton X-100 or NP-40) to effectively solubilize the membrane fraction. Avoid excessive heating of samples (limit to 70°C for 5 minutes) to prevent aggregation of transmembrane proteins.

  2. Gel percentage: Use 10-12% polyacrylamide gels for optimal separation around the 34-35 kDa range where OR4C15 is detected .

  3. Transfer conditions: For transmembrane proteins, use PVDF membranes (rather than nitrocellulose) and add 10-20% methanol to the transfer buffer to improve transfer efficiency.

  4. Blocking: Use 5% non-fat milk or 3-5% BSA in TBST (as specified in product protocols) . For phospho-specific detection, BSA is preferable as milk contains phosphoproteins.

  5. Antibody concentration: Start with the manufacturer's recommended dilution (typically 1:500-1:2000) and optimize as needed. For OR4C15, overnight incubation at 4°C often yields better results than shorter incubations.

  6. Controls: Include positive controls such as HepG2 or Jurkat cell lysates which have been validated for OR4C15 expression . Consider using OR4C15 overexpression systems as additional positive controls.

  7. Detection system: HRP-conjugated secondary antibodies with ECL detection systems are typically sufficient, but for low abundance targets, consider more sensitive detection methods.

• What are the best methods for validating OR4C15 antibody specificity?

  Validating antibody specificity is crucial for reliable research. For OR4C15 antibodies, consider these validation approaches:

  1. Knockout/knockdown controls: Use CRISPR/Cas9 knockout or siRNA knockdown of OR4C15 to demonstrate loss of signal in Western blot or IF applications.

  2. Overexpression systems: Test the antibody on cells transfected with OR4C15 expression plasmids (such as the untagged clone SC300339) versus empty vector controls.

  3. Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to demonstrate signal reduction.

  4. Cross-reactivity testing: Test the antibody on tissues/cells from different species to confirm the specified reactivity pattern (human, rat, mouse as applicable) .

  5. Multiple antibodies comparison: Use antibodies raised against different epitopes of OR4C15 and compare staining patterns.

  6. Mass spectrometry validation: Immunoprecipitate OR4C15 using the antibody and verify the pulled-down protein by mass spectrometry.

  7. Application-specific validations: For IF/ICC, co-stain with markers of expected subcellular localization (membrane proteins). For WB, verify the observed molecular weight matches the expected 34-35 kDa .

• How can I optimize immunofluorescence protocols for detecting OR4C15 in tissue sections?

  For optimal immunofluorescence detection of OR4C15 in tissue sections:

  1. Fixation: Use 4% paraformaldehyde for 15-20 minutes for cell slides, or overnight for tissue sections . For OR4C15, which is a membrane protein, avoid over-fixation which can mask epitopes.

  2. Antigen retrieval: Perform heat-induced antigen retrieval using citric acid solution or microwave treatment . This is particularly important for formalin-fixed tissues to expose the epitopes.

  3. Permeabilization: Use 0.1% Triton X-100 for 10 minutes to ensure antibody access to membrane proteins .

  4. Blocking: Use 1-3% BSA in PBS for 1 hour at room temperature to reduce non-specific binding .

  5. Antibody dilution: For OR4C15 antibodies, use dilutions of 1:100-1:500 for IF applications . For most consistent results, incubate overnight at 4°C.

  6. Secondary antibody: Use species-appropriate fluorescent-conjugated secondary antibodies (anti-rabbit for most OR4C15 antibodies) at manufacturer recommended dilutions.

  7. Nuclear counterstain: Include DAPI (5 minutes at room temperature) to visualize nuclei .

  8. Controls: Include both positive (tissues known to express OR4C15) and negative (primary antibody omission) controls. Consider dual staining with known membrane markers to confirm localization.

  9. Mounting and imaging: Use anti-fade mounting medium and examine using confocal microscopy for optimal resolution of membrane structures .

Advanced Research Questions

• What strategies should be employed when investigating potential OR4C15 interactions with other olfactory signaling proteins?

  Investigating protein-protein interactions involving OR4C15 requires specialized approaches for membrane proteins:

  1. Co-immunoprecipitation (Co-IP): Use mild detergents (0.5-1% digitonin or DDM) to solubilize membrane complexes while maintaining protein-protein interactions. Cross-link proteins before lysis (using DSP or formaldehyde) to stabilize transient interactions. OR4C15 antibodies can be used for IP followed by Western blotting for potential interaction partners.

  2. Proximity ligation assay (PLA): This technique can detect protein interactions in situ with high sensitivity. Use OR4C15 antibodies in combination with antibodies against potential interaction partners, followed by oligonucleotide-linked secondary antibodies that generate a fluorescent signal when in close proximity.

  3. FRET/BRET analysis: Generate fluorescent protein fusions with OR4C15 and candidate interactors to study their interactions in living cells through resonance energy transfer techniques.

  4. Split reporter assays: Use protein complementation assays where OR4C15 and potential partners are fused to complementary fragments of a reporter protein (luciferase or fluorescent protein).

  5. Mammalian two-hybrid system: Particularly useful for membrane proteins like OR4C15, this system can detect interactions in a cellular context more relevant than yeast systems.

  6. Mass spectrometry: After immunoprecipitation with OR4C15 antibodies, identify interacting proteins by mass spectrometry analysis. For transmembrane proteins like OR4C15, specialized sample preparation and analysis may be required.

  7. Functional validation: Confirm the physiological relevance of identified interactions through functional assays such as calcium imaging or cAMP measurements in response to potential odorants.

• How can OR4C15 antibodies be effectively used in conditional reprogramming culture systems for personalized medicine applications?

  Conditional reprogramming (CR) culture systems represent an emerging platform for personalized medicine. OR4C15 antibodies can be integrated into these systems following these methodological considerations:

  1. Cell culture characterization: Use OR4C15 antibodies to characterize expression patterns in patient-derived conditionally reprogrammed cells, particularly when studying olfactory or neuronal lineages. IF/ICC applications at 1:100-1:500 dilutions are optimal for this purpose .

  2. Co-expression analysis: Combine OR4C15 staining with lineage markers (like pan-keratin, CD44) to assess cellular heterogeneity and characterize subpopulations . This helps identify cells that may represent different functional states within the culture.

  3. Drug response monitoring: Implement OR4C15 antibodies in high-content screening workflows after drug treatments. After treating cultured cells with compounds of interest, perform immunofluorescence with OR4C15 antibodies to detect changes in expression or localization that correlate with drug response .

  4. Protocol optimization: For CR cultures specifically, modify standard immunofluorescence protocols to include:

     • Fixation in 4% paraformaldehyde for 15 minutes

     • Permeabilization with 0.1% Triton X-100 for 10 minutes

     • Blocking with 1% BSA for 1 hour

     • Primary antibody incubation at 4°C overnight (OR4C15 antibody at 1:200)

     • DAPI counterstaining for 5 minutes

  5. Organoid applications: For organoid cultures derived through conditional reprogramming, implement whole mount staining methods with OR4C15 antibodies to visualize three-dimensional expression patterns .

  6. Correlation with clinical outcomes: When using CR cultures for personalized medicine, correlate OR4C15 expression patterns with patient clinical data to identify potential biomarkers of treatment response.

• What are the critical considerations when using OR4C15 antibodies in multiplex immunofluorescence assays?

  Multiplex immunofluorescence with OR4C15 antibodies requires careful planning:

  1. Antibody compatibility: When combining OR4C15 antibodies (typically rabbit-derived) with other primary antibodies, select those raised in different host species (mouse, goat, etc.) to allow for species-specific secondary antibodies without cross-reactivity.

  2. Spectral separation: Choose fluorophores with minimal spectral overlap to reduce bleed-through. For OR4C15 detection alongside other targets, consider using fluorophores such as:

     • OR4C15 (rabbit primary): Anti-rabbit Alexa Fluor 488

     • Target 2 (mouse primary): Anti-mouse Alexa Fluor 568

     • Target 3 (goat primary): Anti-goat Alexa Fluor 647

  3. Sequential immunostaining: For multiple rabbit antibodies including OR4C15, use sequential immunostaining with intermediate stripping or blocking steps. Tyramide signal amplification (TSA) methods can be particularly effective here.

  4. Optimization of antibody concentrations: In multiplex settings, individual antibody concentrations often need adjustment. For OR4C15, start with the lower end of the recommended range (e.g., 1:500 for IF) and adjust based on signal intensity .

  5. Controls: Include single-stained controls for each antibody to assess bleed-through, as well as secondary-only controls to evaluate background.

  6. Image acquisition settings: Use sequential scanning on confocal systems rather than simultaneous acquisition to minimize channel crosstalk.

  7. Image analysis: Employ computational methods for spectral unmixing if needed, particularly in tissues with high autofluorescence.

  8. Colocalization assessment: When studying OR4C15 interaction with other proteins, use appropriate colocalization metrics (Pearson's coefficient, Manders' overlap) rather than visual assessment alone.

• How can I troubleshoot non-specific binding or weak signals when using OR4C15 antibodies?

  When encountering issues with OR4C15 antibody performance, consider these troubleshooting strategies:

  For non-specific binding:

  1. Increase blocking stringency: Extend blocking time to 2 hours and increase BSA concentration to 3-5%. Consider adding 5-10% serum from the secondary antibody host species.

  2. Optimize antibody dilution: Use a dilution series to determine the optimal concentration that maximizes specific signal while minimizing background. For OR4C15, this may be higher than the manufacturer's recommendation (try 1:1000-1:3000 for WB) .

  3. Modify washing protocol: Increase the number and duration of wash steps (5-6 washes of 10 minutes each with 0.1% Tween-20 in PBS).

  4. Pre-adsorb antibody: Incubate the diluted antibody with tissues/cells lacking OR4C15 to remove antibodies that bind non-specifically.

  5. Test different blocking agents: If BSA doesn't sufficiently reduce background, try 5% non-fat milk, normal serum, or commercial blocking reagents.

  For weak signals:

  1. Epitope retrieval optimization: For fixed tissues, test different antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0, enzymatic retrieval) as the epitope may be masked .

  2. Increase antibody concentration: Try higher concentrations of primary antibody (1:100-1:200 for IF/ICC) .

  3. Extend incubation time: Incubate primary antibody overnight at 4°C or even up to 48 hours for difficult tissues.

  4. Use signal amplification: Implement tyramide signal amplification (TSA) or polymer-based detection systems to enhance sensitivity.

  5. Check sample preparation: Ensure proper fixation protocols that preserve epitope integrity while allowing antibody access.

  6. Verify target expression: Confirm OR4C15 expression in your sample type with alternative methods (qPCR, RNA-seq data) before troubleshooting antibody conditions.

  7. Try different antibody clones: If available, test OR4C15 antibodies targeting different epitopes as accessibility may vary by application or tissue type.

• What methodologies are recommended for studying OR4C15 expression across different olfactory cell types?

  For comprehensive analysis of OR4C15 expression across olfactory cell types:

  1. Single-cell RNA sequencing: This provides transcriptome-wide data on OR4C15 expression in individual cells, allowing identification of cell-type specific expression patterns. Complement with protein validation using OR4C15 antibodies.

  2. Laser capture microdissection: Isolate specific regions or cell types from olfactory epithelium sections, followed by qPCR for OR4C15 mRNA or Western blot with OR4C15 antibodies for protein detection.

  3. Multiplex immunofluorescence: Combine OR4C15 antibodies with markers for specific olfactory cell types:

     • Mature olfactory sensory neurons (OSNs): OMP (Olfactory Marker Protein)

     • Immature OSNs: GAP43

     • Basal cells: p63, Keratin 5

     • Sustentacular cells: SUS-1, CYP2A5

  4. Flow cytometry: Dissociate olfactory epithelium and perform intracellular staining for OR4C15 alongside surface markers for different cell populations. Use OR4C15 antibodies at approximately 1:100 dilution for this application.

  5. In situ hybridization with immunofluorescence: Combine RNAscope for OR4C15 mRNA with immunofluorescence for cell-type markers to correlate transcript and protein levels.

  6. Tissue clearing techniques: For 3D visualization of OR4C15 distribution in intact olfactory tissues, combine tissue clearing methods (CLARITY, iDISCO) with whole-mount immunostaining using OR4C15 antibodies.

  7. Organoid models: Generate olfactory organoids and use OR4C15 antibodies to study developmental expression patterns in a controlled system.

  8. Transgenic reporter approaches: When possible, use OR4C15 promoter-driven fluorescent reporters in combination with antibody staining for other markers to track expression in living tissue.