OR51F2 Antibody

What is OR51F2 and what cellular functions does it perform?

OR51F2 (Olfactory receptor 51F2, also known as Olfactory receptor OR11-23) is a member of the olfactory receptor family 51 subfamily F. It functions primarily as an odorant receptor and is a multi-pass membrane protein localized to the cell membrane . This protein is encoded by the OR51F2 gene (Gene ID: 119694) and has the UniProt identifier O51F2_HUMAN . Olfactory receptors like OR51F2 are G-protein coupled receptors that detect odor molecules in the nasal epithelium and initiate neuronal responses that trigger smell perception. Understanding OR51F2's cellular distribution and functional properties is fundamental to research in sensory neuroscience and chemosensory signal transduction pathways.

What are the common applications for OR51F2 antibodies in research?

OR51F2 antibodies are utilized across multiple experimental platforms in molecular and cellular biology research. Based on manufacturer specifications, the primary applications include Western Blot (WB), Immunofluorescence (IF), and ELISA . Some antibody variants also support Immunohistochemistry (IHC) and Immunocytochemistry (ICC) applications . These techniques enable researchers to:

• Detect endogenous levels of OR51F2 protein in cell or tissue samples

• Characterize protein expression patterns in different cell types

• Investigate subcellular localization of OR51F2

• Quantify protein levels in experimental conditions

When selecting an OR51F2 antibody, researchers should confirm that it has been validated for their specific application of interest, as performance can vary significantly across different experimental contexts .

What is the molecular weight of OR51F2 and how can this information guide Western blot analysis?

When conducting Western blot analysis with OR51F2 antibodies:

• Expected band size should be approximately 35-39 kDa for the native protein

• Post-translational modifications may cause the actual migration pattern to differ from the calculated weight

• Positive controls such as K562 cells have been validated for Western blot detection of OR51F2

• Sample preparation methods (reducing vs. non-reducing conditions) should be optimized based on the antibody specifications

Researchers should carefully validate band specificity using appropriate controls to distinguish specific OR51F2 signal from potential cross-reactive proteins of similar molecular weight.

How do different epitope regions of OR51F2 antibodies affect experimental outcomes?

The epitope specificity of OR51F2 antibodies significantly impacts experimental performance and data interpretation. Several commercially available antibodies target different regions of the OR51F2 protein:

The selection of antibodies targeting specific epitope regions has several research implications:

Antibodies targeting the 292-341 region (C-terminal domain) may provide better accessibility in applications where the protein maintains its membrane-embedded conformation. This region likely represents an extracellular or cytoplasmic domain that remains accessible even when the protein is in its native conformation .

In contrast, antibodies targeting smaller epitopes (like the 324-336 region) might offer higher specificity but potentially lower sensitivity depending on epitope accessibility . For critical experiments, researchers should consider validating results with multiple antibodies targeting different epitopes to confirm specificity and rule out artifacts.

What cross-reactivity patterns should researchers consider when using OR51F2 antibodies across species?

Species cross-reactivity is a crucial consideration for comparative studies involving OR51F2. The observed reactivity patterns vary across different antibody products:

This cross-reactivity information is critical when designing comparative studies. Researchers should note that:

For definitive cross-species comparisons, researchers should perform preliminary validation using positive control samples from each species of interest and consider epitope sequence conservation analysis .

How can researchers optimize double immunofluorescence staining protocols when using OR51F2 antibodies?

Double immunofluorescence staining with OR51F2 antibodies requires careful optimization to achieve specific labeling while avoiding cross-reactivity and background issues. Based on available antibody conjugates and properties, researchers should consider:

• Conjugate selection: FITC-conjugated OR51F2 antibodies (excitation/emission: 499/515 nm, laser line: 488 nm) are available for direct fluorescence detection . These can be paired with antibodies against other targets conjugated to spectrally distinct fluorophores.

• Sequential staining protocol:

  • Fix samples using 4% paraformaldehyde (10-15 minutes at room temperature)

  • Permeabilize with 0.1-0.3% Triton X-100 if intracellular epitopes are targeted

  • Block with 5-10% normal serum from the species unrelated to both primary antibodies

  • Apply first primary antibody at optimized dilution (typical IF dilution range for OR51F2: 1:200-1:1000)

  • Incubate overnight at 4°C

  • Wash thoroughly (3-5 times with PBS)

  • Apply second primary antibody (from different host species)

  • Apply appropriate secondary antibodies with non-overlapping emission spectra

  • Include rigorous controls (single-antibody stains and secondary-only controls)

• For co-localization studies with membrane proteins, super-resolution microscopy techniques may be necessary to resolve OR51F2's distribution within the cell membrane accurately.

Optimizing the concentration of each antibody is critical, as fluorescence imaging can be particularly sensitive to nonspecific binding. Researchers should initially test dilutions at the upper end of the recommended range (e.g., 1:1000) and titrate to find optimal signal-to-noise ratios .

What are the recommended protocols for Western blot detection of OR51F2?

Optimized Western blot protocols for OR51F2 detection should account for its membrane protein characteristics and molecular weight. Based on validated antibody specifications, the following methodological approach is recommended:

• Sample preparation:

  • Lyse cells in RIPA buffer containing protease inhibitors

  • For membrane proteins like OR51F2, include 1% SDS to ensure complete solubilization

  • Heat samples at 95°C for 5 minutes in reducing Laemmli buffer

• Gel electrophoresis and transfer:

  • Use 10-12% polyacrylamide gels for optimal resolution around 35-39 kDa

  • Transfer to PVDF membrane (preferred over nitrocellulose for hydrophobic proteins)

  • Use wet transfer system at low voltage (30V) overnight at 4°C to ensure complete transfer of membrane proteins

• Antibody incubation:

  • Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with primary anti-OR51F2 antibody at dilution 1:500-1:2000

  • Optimize incubation conditions (overnight at 4°C typically yields best results)

  • Wash thoroughly with TBST (3-5 times, 5 minutes each)

  • Incubate with HRP-conjugated secondary antibody (typically 1:5000-1:10000)

• Detection:

  • Develop using enhanced chemiluminescence (ECL) reagents

  • Expected band size: approximately 35-38.5 kDa

• Validated positive controls:

  • K562 cells have been confirmed to express detectable levels of OR51F2

For quantitative Western blot analysis, researchers should include appropriate loading controls and consider using fluorescent secondary antibodies with digital imaging systems to ensure measurements fall within the linear range of detection.

What immunofluorescence protocol modifications are necessary for optimal OR51F2 detection in different cell types?

Immunofluorescence detection of OR51F2 requires protocol modifications based on the cell type and the specific cellular compartment being investigated. For optimal results:

• Cell type-specific considerations:

  • Epithelial cells: Standard 4% paraformaldehyde fixation (10 minutes) is typically sufficient

  • Neuronal cells: Milder fixation (2% paraformaldehyde, 10 minutes) may better preserve membrane protein epitopes

  • Primary tissues: Consider antigen retrieval methods for formalin-fixed samples

• Permeabilization optimization:

  • For membrane protein OR51F2, minimize permeabilization (0.1% Triton X-100 for 5 minutes)

  • Alternative: Use 0.1% saponin which creates smaller pores that maintain membrane structure

  • For selective plasma membrane labeling, try non-permeabilizing conditions first

• Antibody dilution optimization:

  • Start with 1:200-1:1000 dilution range as recommended

  • Titrate for each cell type to determine optimal signal-to-noise ratio

  • Extended incubation (overnight at 4°C) typically improves specific labeling

• Signal amplification options:

  • Tyramide signal amplification can enhance detection of low-abundance OR51F2

  • Biotin-streptavidin systems provide alternative amplification

• Mounting considerations:

  • Use anti-fade mounting media containing DAPI for nuclear counterstaining

  • For long-term storage, seal edges with nail polish to prevent drying

When comparing OR51F2 expression across different cell types, it's essential to maintain consistent fixation and staining protocols to allow valid quantitative comparisons of fluorescence intensity .

How should researchers validate the specificity of OR51F2 antibodies in their experimental systems?

Rigorous validation of OR51F2 antibody specificity is essential for reliable experimental outcomes. Recommended validation approaches include:

• Positive and negative control samples:

  • Positive controls: K562 cells have been verified to express OR51F2

  • Negative controls: Use cell lines known not to express OR51F2 or tissues from knockout models if available

• Blocking peptide controls:

  • Pre-incubate the antibody with excess immunizing peptide (available as OR51F2 blocking peptide)

  • Run parallel experiments with blocked and unblocked antibody

  • Specific signals should be eliminated in the blocked condition

• Knockdown/knockout verification:

  • Perform siRNA knockdown of OR51F2

  • Compare antibody staining in knockdown vs. control samples

  • Specific signals should be significantly reduced in knockdown samples

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of OR51F2 (e.g., compare results from antibodies targeting regions 292-341 and 324-336)

  • Consistent staining patterns across different antibodies increase confidence in specificity

• Mass spectrometry validation:

  • Immunoprecipitate OR51F2 using the antibody

  • Confirm protein identity by mass spectrometry

  • This provides definitive validation of antibody specificity

• Western blot validation:

  • Verify single band of expected molecular weight (35-39 kDa)

  • Multiple bands or unexpected molecular weights may indicate cross-reactivity

Researchers should document validation results thoroughly and include appropriate validation controls in experimental designs and publications to ensure reproducibility and reliability of findings.

How can researchers design experiments to investigate OR51F2 expression in olfactory versus non-olfactory tissues?

Investigating OR51F2 expression across olfactory and non-olfactory tissues requires careful experimental design that addresses tissue-specific challenges:

• Tissue panel selection:

  • Olfactory tissues: Olfactory epithelium (primary site)

  • Non-olfactory tissues with reported expression: Prostate (high expression), spleen, liver, retinal pigment epithelium, melanocytes, medulla oblongata

  • Negative control tissues: Tissues with minimal expected expression (e.g., skeletal muscle)

• Multi-method expression analysis approach:

  • qRT-PCR to quantify mRNA levels (design primers specific to OR51F2, avoiding closely related family members)

  • Western blot for protein expression (35-39 kDa band)

  • Immunohistochemistry/immunofluorescence for spatial distribution analysis

  • Single-cell RNA-seq for cell-type specific expression patterns

• Olfactory tissue-specific considerations:

  • Fresh olfactory tissue is preferable due to rapid degradation post-mortem

  • Consider specialized fixation protocols (e.g., short fixation times with 2-4% PFA)

  • Use established olfactory epithelium markers (e.g., OMP, NCAM) for co-localization studies

• Comparative quantification methodology:

  • Normalize protein expression to appropriate housekeeping proteins for each tissue type

  • Consider tissue-specific protein extraction optimization for membrane proteins

  • Use digital image analysis software for objective quantification of immunostaining intensity

• Biological replicates and statistical analysis:

  • Include tissue samples from at least 3-5 different individuals

  • Apply appropriate statistical tests based on data distribution (e.g., ANOVA with post-hoc tests)

  • Report both statistical significance and effect sizes

This comprehensive approach enables robust characterization of OR51F2 expression patterns across diverse tissues and can reveal unexpected expression sites beyond the canonical olfactory system, potentially suggesting non-olfactory functions of this receptor .

What considerations are important when designing co-immunoprecipitation experiments to identify OR51F2 binding partners?

Co-immunoprecipitation (Co-IP) of OR51F2 presents unique challenges due to its multi-pass membrane protein structure. Researchers should consider the following design elements:

• Lysis buffer optimization:

  • Use non-denaturing conditions to preserve protein-protein interactions

  • Include 1-2% digitonin or 0.5-1% DDM (n-Dodecyl β-D-maltoside) instead of harsher detergents like SDS

  • Add protease and phosphatase inhibitors freshly before use

  • Consider crosslinking prior to lysis for transient interactions

• Antibody selection strategy:

  • Choose antibodies validated for immunoprecipitation (not all WB/IF-validated antibodies work for IP)

  • Consider using antibodies targeting different epitopes (N-terminal vs. C-terminal)

  • For tagged constructs, commercial anti-tag antibodies often perform more consistently than anti-OR51F2 antibodies

• Experimental controls:

  • IgG control: Use species-matched irrelevant IgG as negative control

  • Input control: Reserve 5-10% of pre-IP lysate

  • Bead-only control: Include sample with beads but no antibody

  • Reciprocal IP: Confirm interactions by IP of the suspected binding partner

• Validation approaches:

  • Perform mass spectrometry analysis of Co-IP samples to identify novel interactions

  • Confirm specific interactions by reverse Co-IP and other methods (proximity ligation assay, FRET)

  • Use increasing stringency washes to distinguish high-affinity from low-affinity interactions

• Specific considerations for G-protein coupled receptors like OR51F2:

  • Consider using stabilizing agents like GDP or GTPγS to capture specific conformational states

  • For G-protein coupling studies, focus on detecting Gα subunits in Co-IP fractions

  • Include membrane fractionation steps to enrich for membrane-associated complexes

When reporting Co-IP results, researchers should clearly specify the experimental conditions, detergent concentrations, and antibody concentrations used, as these factors significantly impact the protein interactions observed .

How should researchers design experiments to assess the functional activity of OR51F2 in response to potential ligands?

Designing functional assays for OR51F2 activation requires specialized approaches that can detect olfactory receptor signaling. Researchers should consider these methodological strategies:

• Expression system selection:

  • Heterologous expression systems: HEK293 cells are commonly used for olfactory receptor functional studies

  • Establish stable cell lines expressing OR51F2 using lentiviral transduction or selection markers

  • Consider co-expression with receptor trafficking proteins (e.g., RTP1S, Ric8b) to improve surface expression

• Functional readout methodologies:

  • Calcium imaging: Use calcium-sensitive dyes (Fluo-4) or genetically encoded calcium indicators (GCaMP)

  • cAMP assays: Measure cAMP production using FRET-based sensors or ELISA-based detection

  • β-arrestin recruitment assays: Use bioluminescence resonance energy transfer (BRET) to measure receptor activation

  • Electrophysiology: Patch-clamp recording in expressing cells

• Ligand screening approach:

  • Start with known odorants that activate related olfactory receptors

  • Design concentration-response experiments (typically 10⁻⁹ to 10⁻³ M)

  • Include positive controls (e.g., forskolin for cAMP assays) and vehicle controls

  • Consider high-throughput screening approaches for large odorant libraries

• Data analysis and quantification:

  • Calculate EC₅₀ values for active ligands

  • Determine efficacy (maximum response) relative to reference standards

  • Apply appropriate statistical tests for comparing responses across ligands

• Validation strategies:

  • Confirm specificity using cells expressing closely related receptors

  • Use receptor antagonists or competitive ligands when available

  • Perform structure-activity relationship analysis with chemically related ligands

  • Validate findings in native tissues where OR51F2 is expressed

This experimental framework enables systematic characterization of OR51F2 ligand preferences and signaling properties, contributing to understanding both its olfactory functions and potential non-olfactory roles in tissues like prostate and retinal pigment epithelium where it is also expressed .

How can researchers overcome common challenges in Western blot detection of OR51F2?

Western blot detection of OR51F2 can present several technical challenges that researchers should anticipate and address:

• Low signal intensity issues:

  • Increase protein loading (50-100 μg total protein per lane)

  • Reduce antibody dilution (try 1:500 instead of 1:2000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use enhanced detection systems (high-sensitivity ECL reagents)

  • Try different antibodies targeting alternative epitopes

• Multiple bands or non-specific binding:

  • Increase blocking stringency (5% BSA instead of milk)

  • Add 0.1% Tween-20 to antibody dilution buffer

  • Increase washing duration and frequency (5 x 10 minutes)

  • Perform antibody validation with blocking peptide

  • Try antibodies targeting different epitopes to confirm specific bands

• Membrane protein-specific issues:

  • Ensure complete solubilization of membrane fractions

  • Avoid boiling samples (heat to 70°C for 10 minutes instead)

  • Use PVDF membranes instead of nitrocellulose

  • Add 6M urea to sample buffer for highly hydrophobic proteins

  • Consider blue-native PAGE for complex membrane proteins

• Degradation issues:

  • Use fresh samples whenever possible

  • Add additional protease inhibitors to lysis buffer

  • Maintain cold chain throughout sample preparation

  • Avoid freeze-thaw cycles of protein samples

• Molecular weight discrepancies:

  • OR51F2 calculated molecular weight is 38.5 kDa, but may run at approximately 35 kDa

  • Post-translational modifications can cause migration differences

  • Consider deglycosylation experiments if glycosylation is suspected

• Tissue-specific considerations:

  • For prostate tissue (high OR51F2 expression), optimize protein extraction with specialized buffers

  • For retinal pigment epithelium, consider specialized extraction protocols for pigmented tissues

Systematically testing these modifications while changing only one variable at a time will help identify the optimal Western blot conditions for OR51F2 detection in specific experimental contexts.

What strategies can address poor immunofluorescence staining patterns when working with OR51F2 antibodies?

Poor immunofluorescence staining with OR51F2 antibodies can result from multiple factors. The following troubleshooting strategies address common issues:

• Weak or absent signal:

  • Optimize fixation (test 2% vs 4% paraformaldehyde, methanol fixation)

  • Try antigen retrieval methods (citrate buffer pH 6.0, microwave heating)

  • Reduce antibody dilution (start at 1:200 instead of 1:1000)

  • Extend primary antibody incubation (overnight at 4°C or longer)

  • Implement signal amplification (tyramide signal amplification, biotin-streptavidin systems)

  • Try FITC-conjugated direct detection antibodies

• High background or non-specific staining:

  • Increase blocking stringency (5-10% normal serum, add 0.1-0.3% Triton X-100)

  • Pre-absorb antibody with acetone powder from negative control tissues

  • Include additional blocking agents (0.1-1% BSA, 0.1% gelatin)

  • Increase washing duration and frequency (5 x 10 minutes with gentle agitation)

  • Use directly conjugated antibodies to eliminate secondary antibody background

• Membrane protein-specific optimization:

  • Reduce permeabilization stringency to preserve membrane structure

  • Try mild detergents (0.01% saponin or digitonin instead of Triton X-100)

  • For selective surface labeling, perform staining before permeabilization

  • Consider live-cell surface staining for native conformation antigens

• Autofluorescence reduction:

  • Include quenching steps (0.1-1% sodium borohydride for 5 minutes)

  • For tissues with high autofluorescence, use Sudan Black B (0.1-0.3% in 70% ethanol)

  • Consider confocal microscopy with spectral unmixing

  • Use fluorophores with emission spectra distinct from tissue autofluorescence

• Cell type-specific optimization:

  • For neuronal tissues, reduce fixation time and concentration

  • For highly pigmented cells (e.g., retinal pigment epithelium), include bleaching steps

  • For prostate tissue, optimize antigen retrieval conditions

• Controls for troubleshooting:

  • Include known positive control samples (K562 cells)

  • Perform parallel staining with different OR51F2 antibodies targeting distinct epitopes

  • Include blocking peptide controls to verify specificity

Systematic documentation of these optimization steps will help establish reliable immunofluorescence protocols for OR51F2 detection across different experimental systems.

How can researchers resolve discrepancies in OR51F2 detection across different experimental techniques?

When researchers encounter discrepancies in OR51F2 detection between techniques (e.g., positive Western blot but negative immunofluorescence), a systematic troubleshooting approach is required:

• Epitope accessibility considerations:

  • Different techniques expose different epitopes (denatured in WB, native in IF)

  • Try antibodies targeting different regions of OR51F2

  • Consider native vs. denatured Western blot conditions

  • For IF, test various fixation and permeabilization protocols to optimize epitope exposure

• Expression level threshold differences:

  • Western blot may detect lower expression levels than IF due to concentration of protein

  • Implement more sensitive detection methods for low-abundance expression:

    • For WB: Use high-sensitivity chemiluminescent substrates

    • For IF: Try signal amplification systems or more sensitive microscopy (confocal, super-resolution)

• Sample preparation validation:

  • Ensure identical sample sources are used across techniques

  • For cell lines, confirm passage number and culture conditions are consistent

  • For tissue samples, use adjacent sections for different techniques

  • Consider protein extraction efficiency differences between preparations

• Antibody validation across platforms:

  • Not all antibodies perform equally across applications

  • Verify antibody validation data for each specific application

  • Test multiple antibodies in parallel across techniques

  • Consider using tagged OR51F2 constructs with anti-tag antibodies as controls

• Quantitative comparison approach:

  • Develop calibration curves using control samples with known expression levels

  • Use digital image analysis tools for precise quantification of signal intensity

  • Apply statistical analysis to determine significance of observed differences

• Complementary technique integration:

  • Supplement protein detection with mRNA analysis (qRT-PCR, in situ hybridization)

  • Consider orthogonal approaches (mass spectrometry, flow cytometry)

  • For conclusive validation, use genetic approaches (CRISPR knockout, siRNA)

When reporting results with technique discrepancies, researchers should transparently discuss potential methodological limitations and provide a comprehensive technical description of protocols to allow proper assessment by the scientific community .

What future research directions are emerging for OR51F2 antibody applications?

Emerging research directions for OR51F2 antibody applications span several innovative areas that extend beyond traditional detection methods:

• Single-cell analysis applications:

  • Integration with single-cell RNA-seq to correlate protein and mRNA levels at single-cell resolution

  • Mass cytometry (CyTOF) applications for high-dimensional protein profiling

  • Super-resolution microscopy for nanoscale localization of OR51F2 in specialized membrane domains

• Functional genomics integration:

  • Combining CRISPR-Cas9 genome editing with antibody-based detection for precise structure-function studies

  • Optogenetic approaches paired with OR51F2 antibody labeling to correlate activation with localization

  • Proximity labeling approaches (BioID, APEX) to map the OR51F2 protein interaction network

• Clinical research applications:

  • Development of diagnostic applications in prostate cancer research, where OR51F2 shows high expression

  • Investigation of OR51F2 in retinal pigment epithelium disorders

  • Exploration of roles in melanocytes and potential connections to pigmentation disorders

• Technical innovations:

  • Development of intrabodies (intracellular antibodies) for live-cell tracking of OR51F2

  • Nanobody development for improved access to conformational epitopes

  • Bispecific antibodies targeting OR51F2 and interacting proteins for co-localization studies

• Comparative biology studies:

  • Cross-species investigation of OR51F2 homologs using antibodies with validated cross-reactivity

  • Evolutionary studies of olfactory receptor expression and function across species

  • Exploration of tissue-specific functions beyond olfaction in different species