OR51A4 Antibody

What is OR51A4 and what is its biological significance?

Olfactory receptor 51A4 (OR51A4) is a member of the olfactory receptor family, which constitutes the largest gene family in the human genome. It functions as a G-protein-coupled receptor (GPCR) with a characteristic 7-transmembrane domain structure common to many neurotransmitter and hormone receptors. OR51A4 plays a critical role in the olfactory system by interacting with specific odorant molecules in the nasal epithelium, initiating neuronal responses that trigger smell perception through G protein-mediated signal transduction pathways .

The protein is encoded by a single exon gene and is part of the OR51 subfamily located on chromosome 11. Understanding OR51A4 expression and function contributes to our knowledge of olfactory coding, sensory perception mechanisms, and potentially certain smell disorders .

What applications are OR51A4 antibodies validated for?

Commercial OR51A4 antibodies are typically validated for multiple applications:

While these applications represent the standard validated uses, researchers should always perform preliminary optimization experiments for their specific tissue or cell types of interest, as expression levels and accessibility of epitopes may vary significantly between experimental systems .

What is the expected molecular weight of OR51A4 in Western blot applications?

There is a notable discrepancy in the literature regarding the molecular weight of OR51A4:

• Calculated molecular weight: ~35 kDa based on amino acid sequence

• Observed molecular weight: ~72 kDa in some experimental systems

This discrepancy is likely due to post-translational modifications such as glycosylation, which is common in membrane proteins, particularly GPCRs. Additionally, the hydrophobic nature of the seven transmembrane domains can affect protein migration in SDS-PAGE. When performing Western blot analysis, researchers should be prepared to observe bands at both the theoretical 35 kDa size and potentially at higher molecular weights. Consider using positive control samples with confirmed OR51A4 expression to establish the expected band pattern in your experimental system .

What are the optimal storage conditions for OR51A4 antibodies?

For long-term storage of OR51A4 antibodies, the consensus recommendation is:

• Store at -20°C for up to one year from receipt

• For short-term storage and frequent use, 4°C for up to one month is acceptable

• Avoid repeated freeze-thaw cycles as this can lead to antibody degradation and loss of activity

Most commercial OR51A4 antibodies are supplied in a buffer containing PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide . This formulation helps maintain stability during storage. Always centrifuge the antibody briefly before opening the vial to collect solution at the bottom, and consider aliquoting the antibody upon first thaw to minimize freeze-thaw cycles if frequent use is anticipated .

How should I optimize dilutions for different experimental applications?

The following methodological approach is recommended for dilution optimization:

• Western Blot Optimization:

  • Start with a 1:1000 dilution in 5% BSA or milk blocking buffer

  • Perform a gradient experiment (e.g., 1:500, 1:1000, 1:2000) if signal strength is inadequate

  • Include a negative control sample without OR51A4 expression

  • Incubate primary antibody overnight at 4°C for optimal results

• Immunofluorescence Optimization:

  • Begin with 1:200 dilution for tissue sections and 1:500 for cultured cells

  • Adjust fixation methods (4% paraformaldehyde is standard for membrane proteins)

  • Consider antigen retrieval methods for tissue sections

  • Include proper blocking (5-10% normal serum) to reduce background

  • Extend primary antibody incubation to overnight at 4°C

• ELISA Protocol:

  • The high dilution (1:10000) reflects the high sensitivity of ELISA

  • Perform a checkerboard titration with both antigen and antibody dilutions

  • Include standard curves using recombinant OR51A4 protein if quantification is needed

Document all optimization steps methodically to establish reproducible protocols specific to your experimental system .

What is the immunogen sequence used for commercial OR51A4 antibodies?

Most commercial OR51A4 antibodies are generated using an immunogen corresponding to amino acids 200-249 of the human OR51A4 protein . This region was selected based on several criteria:

• Surface accessibility based on protein structure prediction

• Unique sequence compared to other olfactory receptors

• Hydrophilic character suitable for antibody recognition

• Low sequence conservation across species (when human-specific detection is desired)

Understanding the exact immunogen region is crucial for:

• Predicting potential cross-reactivity with other olfactory receptors

• Interpreting negative results in cases where that epitope might be masked

• Designing blocking peptides for specificity validation

• Explaining potential differences in antibody performance across applications

When choosing between different commercial antibodies, comparing immunogen sequences can help select the most appropriate reagent for specific experimental needs .

How does OR51A4 copy number variation affect antibody detection and experimental design?

The OR51A4 gene exhibits copy number variation (CNV) in human populations, with structural variations that can significantly impact experimental design and interpretation:

Copy number variations documented in human OR51A4 include:

• Deletions that can remove the entire OR51A4 gene in some individuals

• Complex structural rearrangements in the olfactory receptor gene cluster

Methodological implications for antibody-based studies:

• Population Considerations:

  • Include adequate sample sizes to account for CNV-related expression differences

  • Consider genotyping subjects for OR51A4 CNV status when conducting expression studies

• Control Selection:

  • Use cell lines with confirmed OR51A4 copy number status as controls

  • Include multiple control samples to account for natural variation

• Data Interpretation:

  • Absence of signal may indicate CNV-related absence of the gene rather than technical failure

  • Quantitative differences between samples may reflect copy number differences rather than biological regulation

• Verification Methods:

  • Complement antibody-based detection with genomic PCR to confirm gene presence

  • Consider qPCR to assess copy number when quantitative comparisons are important

These considerations are particularly important in population studies or when using primary human tissues where genetic variation is expected .

What are best practices for validating OR51A4 antibody specificity?

Rigorous validation is essential for confident interpretation of OR51A4 antibody results. A comprehensive validation approach includes:

• Peptide Competition Assay:

  • Pre-incubate the OR51A4 antibody with excess immunogen peptide (aa 200-249)

  • Run parallel experiments with blocked and unblocked antibody

  • Specific signals should disappear in the blocked condition

• Molecular Validation:

  • Use siRNA/shRNA knockdown of OR51A4 in an expressing cell line

  • Compare signal in control vs. knockdown samples

  • Signal reduction should correlate with knockdown efficiency

• Heterologous Expression:

  • Overexpress tagged OR51A4 in a non-expressing cell line

  • Verify co-localization of antibody signal with tag-specific antibodies

  • Confirm signal absence in non-transfected cells

• Multiple Antibody Comparison:

  • Use OR51A4 antibodies from different vendors or raised against different epitopes

  • Compare staining/banding patterns

  • Consistent patterns increase confidence in specificity

• Cross-Reactivity Assessment:

  • Test antibody against closely related olfactory receptors

  • Particularly important for OR51A subfamily members

  • Consider Western blot of lysates from cells expressing different OR51A family members

Documentation of these validation steps should be included in publications to support result interpretation .

How can I optimize immunofluorescence protocols for OR51A4 detection in olfactory tissues?

Detecting OR51A4 in native olfactory tissues presents unique challenges due to low expression levels and the hydrophobic nature of the protein. This optimized protocol addresses these challenges:

• Tissue Preparation:

  • Fix freshly harvested olfactory epithelium in 4% paraformaldehyde (12-24 hours)

  • Consider specialized fixatives containing glutaraldehyde (0.1-0.5%) to better preserve membrane proteins

  • Use sucrose gradient cryoprotection (15%-30%) before freezing

  • Prepare thin sections (8-12 μm) for optimal antibody penetration

• Antigen Retrieval:

  • Heat-mediated antigen retrieval in citrate buffer (pH 6.0) at 95°C for 15-20 minutes

  • Allow slow cooling to room temperature

  • Alternative: sodium borohydride treatment (0.1% for 10 minutes) to reduce aldehyde-induced autofluorescence

• Blocking and Permeabilization:

  • Block with 10% normal serum from secondary antibody host species

  • Add 0.1-0.3% Triton X-100 for membrane permeabilization

  • Include 0.1% saponin to enhance access to transmembrane proteins

  • Extended blocking (2-4 hours) to reduce background

• Antibody Incubation:

  • Dilute primary antibody 1:100-1:500 in blocking solution

  • Incubate for 48-72 hours at 4°C with gentle agitation

  • Multiple washing steps (5-6 changes) with PBS containing 0.1% Tween-20

  • Secondary antibody incubation at 1:500 for 2 hours at room temperature

• Signal Enhancement:

  • Consider tyramide signal amplification for low abundance receptors

  • Use high-sensitivity detection systems (e.g., Quantum Dots or newer fluorophores)

  • Counterstain with DAPI and specific markers for olfactory sensory neurons

• Advanced Imaging:

  • Use confocal microscopy with Z-stack acquisition

  • Apply deconvolution algorithms to enhance signal-to-noise ratio

  • Consider super-resolution techniques for detailed subcellular localization

This protocol may require further optimization based on specific tissue sources and fixation conditions .

What challenges exist in detecting post-translational modifications of OR51A4?

OR51A4, like other GPCRs, undergoes multiple post-translational modifications that can affect antibody recognition and protein function. Key considerations include:

• N-linked Glycosylation:

  • Contributes to the discrepancy between calculated (35 kDa) and observed (72 kDa) molecular weights

  • Can mask epitopes, particularly in the N-terminal extracellular domain

  • Methodological approach: Treat samples with PNGase F to remove N-linked glycans before Western blot

  • Compare migration patterns before and after deglycosylation

• Phosphorylation:

  • Occurs primarily on intracellular loops and C-terminal domain

  • Regulates receptor desensitization and internalization

  • Detection method: Use phospho-specific antibodies or phospho-protein enrichment before analysis

  • Phosphatase inhibitors must be included in all extraction buffers

• Palmitoylation:

  • Common GPCR modification affecting membrane association

  • Can alter detergent solubility during extraction

  • Approach: Compare hydroxylamine-treated vs. untreated samples to identify palmitoylation

  • Modified extraction protocols with appropriate detergents may be required

• Ubiquitination:

  • Regulates receptor degradation and trafficking

  • Detection: Immunoprecipitate OR51A4 and probe with anti-ubiquitin antibodies

  • Consider proteasome inhibitors in cell culture experiments

These modifications affect not only detection but also biological function. Researchers investigating OR51A4 signaling should consider how these modifications might change during receptor activation and desensitization cycles .

How can OR51A4 antibodies be used to study GPCR trafficking and internalization?

OR51A4 antibodies can be powerful tools for investigating the dynamic processes of GPCR trafficking, internalization, and recycling. A methodological framework includes:

• Surface Receptor Quantification:

  • Cell surface biotinylation followed by streptavidin pull-down and Western blot

  • Flow cytometry of non-permeabilized cells using antibodies against extracellular domains

  • Surface ELISA using antibodies against extracellular epitopes

• Internalization Assays:

  • Live-cell antibody feeding: Incubate live cells with antibody at 4°C, then warm to 37°C with/without agonist

  • Track antibody-receptor complexes using fluorescently-labeled secondary antibodies

  • Quantify internalization rates by acid wash to remove surface-bound antibody

  • Time-course analysis using confocal microscopy and co-localization with endosomal markers

• Recycling Studies:

  • Pulse-chase protocols with antibody labeling

  • Double-labeling with different fluorophores to distinguish newly recycled from persistent surface receptors

  • Co-localization with Rab GTPases that mark different recycling compartments

• Advanced Imaging Approaches:

  • FRAP (Fluorescence Recovery After Photobleaching) to analyze lateral mobility

  • TIRF (Total Internal Reflection Fluorescence) microscopy to visualize membrane-proximal events

  • Single-particle tracking of antibody-labeled receptors

• Biochemical Fractionation:

  • Density gradient separation of cellular compartments

  • Immunoblotting fractions to detect OR51A4 distribution

  • Co-immunoprecipitation to identify trafficking-related binding partners

These methods can be applied to study how odorant binding affects OR51A4 trafficking, providing insights into olfactory adaptation mechanisms at the molecular level .

Why might Western blot for OR51A4 show multiple bands or unexpected molecular weights?

Multiple bands or unexpected molecular weights in OR51A4 Western blots can result from several biological and technical factors:

For optimal results, include both positive and negative controls with each Western blot experiment. The positive control should be a sample known to express OR51A4 (e.g., olfactory epithelium lysate), while a negative control could be tissue known not to express olfactory receptors or samples from OR51A4 knockout models if available .

How do I interpret conflicting OR51A4 antibody results across different experimental systems?

When faced with conflicting results using OR51A4 antibodies across different experimental systems, consider this systematic approach to interpretation:

This systematic approach not only helps resolve conflicting results but also advances methodological understanding for the broader research community .

What strategies can improve detection of low-abundance OR51A4 in non-olfactory tissues?

Recent studies suggest OR51A4 may be expressed at low levels in non-olfactory tissues, presenting detection challenges. These advanced strategies can improve sensitivity:

• Sample Enrichment Techniques:

  • Membrane protein fractionation to concentrate GPCRs

  • Lectin affinity purification to enrich glycosylated receptors

  • Immunoprecipitation before Western blot analysis

  • Proximity ligation assay (PLA) for in situ detection of low-abundance proteins

• Signal Amplification Methods:

  • Tyramide signal amplification (TSA) for immunohistochemistry/immunofluorescence

  • Polymer-based detection systems with multiple secondary antibodies

  • Rolling circle amplification for massively enhanced sensitivity

  • Quantum dots as fluorescent labels for improved signal-to-noise ratio

• Specialized Extraction Protocols:

  • Optimize detergent composition for GPCR solubilization (CHAPS, DDM, or digitonin)

  • Include receptor-stabilizing agents (cholesteryl hemisuccinate)

  • Use specialized GPCR extraction kits with optimized buffer systems

  • Perform extractions at 4°C with extended solubilization times

• Advanced Detection Systems:

  • Digital droplet Western blot for single-molecule sensitivity

  • Microwestern arrays for higher sensitivity than conventional Western blot

  • Single-molecule array (Simoa) technology for ultra-sensitive protein detection

  • Mass spectrometry with targeted multiple reaction monitoring (MRM)

• Complementary Approaches:

  • RNAscope in situ hybridization for sensitive mRNA detection

  • CRISPR-based tagging of endogenous OR51A4 with bright fluorescent proteins

  • High-sensitivity RT-qPCR with pre-amplification steps

  • Single-cell analysis to identify rare expressing cells within tissues

These approaches significantly extend detection limits beyond standard methods and are particularly valuable when investigating potential non-canonical functions of OR51A4 outside the olfactory system .

How can OR51A4 antibodies contribute to research on olfactory disorders?

OR51A4 antibodies offer valuable tools for investigating various olfactory disorders through multiple research approaches:

• Comparative Expression Analysis:

  • Compare OR51A4 expression between healthy and pathological olfactory epithelium

  • Quantify receptor abundance changes in aging-related olfactory decline

  • Assess receptor distribution alterations in post-viral olfactory dysfunction

  • Methodological approach: Standardized immunohistochemistry with digital quantification

• Receptor Trafficking in Disease States:

  • Examine OR51A4 localization in models of inflammatory olfactory disorders

  • Track receptor internalization rates in response to persistent odorant exposure

  • Investigate recycling defects in neurodegenerative conditions

  • Approach: Live-cell imaging with pulse-chase antibody labeling

• Structural Studies:

  • Use antibodies for immunoprecipitation of native receptor for structural analysis

  • Investigate conformational changes in disease-associated receptor variants

  • Study protein-protein interactions in health vs. disease states

  • Methodology: Crosslinking immunoprecipitation followed by mass spectrometry

• Diagnostic Applications:

  • Develop antibody panels including OR51A4 for olfactory biopsy analysis

  • Establish quantitative benchmarks for receptor expression in healthy tissue

  • Correlate receptor alterations with specific olfactory dysfunction patterns

  • Approach: Multiplexed immunofluorescence with automated image analysis

These approaches contribute to understanding mechanisms underlying conditions such as congenital anosmia, post-viral smell loss, and neurodegenerative-associated olfactory dysfunction, potentially informing future therapeutic strategies .

What are the emerging applications of OR51A4 antibodies in neuroscience research?

Beyond traditional olfactory studies, OR51A4 antibodies are enabling several innovative research directions in neuroscience:

• Ectopic Expression Studies:

  • Investigation of OR51A4 expression in non-olfactory neurons

  • Characterization of potential chemosensory roles in other neural systems

  • Methodological approach: High-sensitivity multiplexed immunohistochemistry with neuronal subtype markers

  • Application: Understanding broader chemosensory mechanisms throughout the nervous system

• Neural Circuit Mapping:

  • Tracing connections of OR51A4-expressing olfactory sensory neurons

  • Correlating receptor expression with specific glomerular targets

  • Method: Combined immunolabeling with anterograde/retrograde tracers

  • Significance: Constructing precise olfactory coding maps for specific odorant reception

• Developmental Neurobiology:

  • Tracking OR51A4 expression during olfactory system development

  • Correlating receptor expression with axon guidance and targeting

  • Approach: Temporal analysis using immunohistochemistry at different developmental stages

  • Impact: Understanding mechanisms of olfactory circuit formation and refinement

• Neuroplasticity Research:

  • Monitoring OR51A4 expression changes following olfactory training or deprivation

  • Quantifying receptor dynamics during regeneration after injury

  • Methodology: Longitudinal studies with standardized quantitative immunofluorescence

  • Application: Developing strategies to promote olfactory system recovery and adaptation

• Single-Cell Heterogeneity:

  • Examining OR51A4 co-expression with other signaling molecules at single-cell resolution

  • Investigating receptor mosaicism across the olfactory epithelium

  • Method: Multiplexed immunofluorescence combined with computational spatial analysis

  • Significance: Understanding the complexity of olfactory coding at cellular resolution

These emerging applications significantly expand the utility of OR51A4 antibodies beyond traditional protein detection to address fundamental questions in neuroscience research .