OR5AU1 Antibody

What is OR5AU1 and what is its biological function?

OR5AU1 (olfactory receptor family 5 subfamily AU member 1) is a protein-coding gene located on chromosome 14 in humans. It belongs to the large family of G-protein-coupled receptors (GPCRs) involved in olfactory perception. The OR5AU1 protein functions as an odorant receptor, interacting with odorant molecules in the nose to initiate neuronal responses that trigger smell perception .

Functionally, OR5AU1 shares a characteristic 7-transmembrane domain structure with many neurotransmitter and hormone receptors. It is responsible for the recognition and G protein-mediated transduction of odorant signals. The protein is classified as a cell membrane multi-pass membrane protein, localizing to the plasma membrane .

The gene is also known by the synonym OR14-38, and the olfactory receptor gene family is notably the largest gene family in the human genome. The nomenclature assigned to olfactory receptor genes and proteins is species-specific and independent of other organisms .

The Human Protein Atlas data indicates that OR5AU1 expression follows a tissue-specific pattern characteristic of olfactory receptors . When designing experiments involving OR5AU1, researchers should consider:

• Tissue selection: Despite being an olfactory receptor, expression may not be limited to nasal epithelium, as some olfactory receptors have been detected in non-olfactory tissues.

• Positive controls: When validating antibody specificity, use tissues or cell lines with confirmed OR5AU1 expression.

• Negative controls: Include tissues known not to express OR5AU1 or use blocking peptides to verify specificity.

Methodologically, researchers should perform RNA-level validation (e.g., RT-PCR) alongside protein detection to confirm expression patterns, particularly when investigating novel expression sites for OR5AU1 .

How can researchers validate the specificity of OR5AU1 antibodies?

Validating OR5AU1 antibody specificity is critical for reliable research outcomes. A comprehensive validation approach should include:

• Peptide competition assays: Block the antibody with the immunizing peptide prior to application. The signal should be significantly reduced or eliminated, as demonstrated in validation studies where the lane blocked with synthesized peptide shows no signal .

• Knockout/knockdown controls: Use CRISPR/Cas9 or siRNA to generate OR5AU1-deficient samples as negative controls.

• Multiple antibody validation: Use antibodies from different sources or those recognizing different epitopes of OR5AU1.

• Cross-species reactivity assessment: Test the antibody against OR5AU1 from different species to evaluate conservation of epitope recognition.

• Multiple detection methods: Validate findings across different techniques (WB, IF, IHC) to build confidence in specificity.

The search results show that commercial OR5AU1 antibodies typically use synthetic peptides derived from specific amino acid regions (e.g., amino acids 201-250) of human OR5AU1 as immunogens , and validation includes blocking experiments with the immunizing peptide to confirm specificity.

What are the technical challenges in working with olfactory receptor antibodies like OR5AU1?

Researchers face several technical challenges when working with OR5AU1 and other olfactory receptor antibodies:

• High sequence homology: Olfactory receptors comprise a large gene family with significant sequence similarity, complicating antibody specificity. OR5AU1 antibodies may cross-react with related olfactory receptors.

• Membrane protein extraction: As a 7-transmembrane GPCR, OR5AU1 requires careful extraction protocols to maintain native conformation and prevent aggregation.

• Low expression levels: Olfactory receptors often have low expression outside their primary tissues, requiring sensitive detection methods.

• Post-translational modifications: Changes such as glycosylation may affect antibody recognition.

• Fixation sensitivity: For immunohistochemistry and immunofluorescence, fixation conditions can impact epitope availability.

Methodologically, researchers should optimize protein extraction using specialized buffers for membrane proteins, consider using detergents like CHAPS or DDM to solubilize the receptor, and employ signal amplification techniques for detection of low-abundance expression .

What is the evidence for OR5AU1 involvement in non-olfactory tissues and disease states?

While OR5AU1 is primarily an olfactory receptor, research on related olfactory receptors suggests potential roles beyond smell perception:

• Cancer associations: Research on the related olfactory receptor OR51J1 has revealed its potential as a cancer-associated biomarker, showing significantly higher expression in breast tumors compared to normal tissues (2.91-fold increase) . While specific studies on OR5AU1 in cancer weren't prominent in the search results, the finding that some olfactory receptors have altered expression in cancer suggests potential research directions for OR5AU1.

• Genetic disease associations: The search results mentioned OR5AU1 in a table of genes with presumed loss-of-function variants in probands with retinal dystrophy, with a binomial p-value of 4.21 × 10^-4 . This suggests a potential association that warrants further investigation.

For methodological considerations, researchers investigating OR5AU1 in disease contexts should:

• Use quantitative techniques like qRT-PCR to assess expression changes

• Perform immunohistochemistry on tissue microarrays to evaluate protein expression across multiple samples

• Correlate expression with clinical parameters to assess prognostic value

• Consider functional studies to elucidate potential mechanisms beyond olfaction

How can researchers optimize immunohistochemistry (IHC) protocols for OR5AU1 detection?

Based on methodologies described for related olfactory receptors and membrane proteins, researchers should consider the following protocol optimizations for OR5AU1 IHC:

• Tissue preparation and fixation:

  • Use fresh frozen tissue or carefully fixed paraffin-embedded samples

  • Consider shorter fixation times (4-24 hours) with 10% neutral buffered formalin

  • For antigen retrieval, test both heat-induced epitope retrieval (HIER) methods in citrate buffer (pH 6.0) and EDTA buffer (pH 9.0)

• Antibody optimization:

  • Perform antibody titration (1:100-1:1000) to determine optimal concentration

  • Include long primary antibody incubation (overnight at 4°C)

  • Test signal amplification systems like tyramide signal amplification for low-abundance targets

• Controls:

  • Include peptide competition controls by pre-incubating antibody with immunizing peptide

  • Use tissues with known OR5AU1 expression as positive controls

  • Replace primary antibody with PBS as negative controls

• Signal development and quantification:

  • For chromogenic detection, counterstain with hematoxylin and mount in Entellan or similar medium

  • Evaluate staining in different cellular compartments (membrane vs. cytoplasmic)

  • Consider scoring systems similar to those used for other olfactory receptors (negative, 1+, 2+, 3+ for weak, moderate, and strong expression)

What approaches can be used to design antibodies with improved specificity for OR5AU1?

Recent advances in antibody design can be applied to develop more specific OR5AU1 antibodies:

• Epitope selection strategies:

  • Target regions unique to OR5AU1 that have minimal sequence homology with other olfactory receptors

  • Focus on extracellular loops rather than transmembrane domains for improved accessibility

  • Utilize bioinformatic tools to identify OR5AU1-specific epitopes with high antigenicity scores

• Advanced antibody engineering:

  • Apply computational models like those described for designing antibodies with customized specificity profiles

  • Generate high-throughput sequencing data from phage display experiments to identify binding modes specific to OR5AU1

  • Optimize antibodies for either specific high affinity for OR5AU1 or controlled cross-specificity depending on research needs

• Validation approaches:

  • Implement cross-adsorption against related olfactory receptors

  • Use surface plasmon resonance (SPR) to quantify binding kinetics and assess specificity

  • Perform comprehensive cross-reactivity testing against structurally similar olfactory receptors

As described in the research on antibody specificity inference, biophysics-informed modeling combined with selection experiments can be powerful for designing proteins with desired physical properties, including antibodies with tailored specificity profiles .

What are common troubleshooting strategies for Western blot detection of OR5AU1?

Researchers encountering difficulties with OR5AU1 Western blots should consider these methodological approaches:

• Sample preparation issues:

  • Ensure complete solubilization of membrane proteins using appropriate detergents (CHAPS, DDM, or Triton X-100)

  • Avoid sample heating above 70°C, which can cause GPCR aggregation

  • Include protease inhibitors to prevent degradation

• Detection problems:

  • For weak signals, increase protein loading (50-100 μg) or use signal enhancement systems

  • Optimize primary antibody concentration (test range from 1:500-1:2000)

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

  • Test multiple secondary antibody systems (HRP, AP, or fluorescent-based)

• Specificity concerns:

  • Run parallel blots with the antibody pre-incubated with blocking peptide

  • Include positive control lysates such as K562 or Jurkat cells

  • Verify protein size matches the expected 35 kDa molecular weight

• Background reduction:

  • Increase blocking time or concentration (5% BSA or milk)

  • Add 0.05-0.1% Tween-20 in wash buffers

  • Consider using specialized membrane blocking solutions for GPCRs

Commercial OR5AU1 antibodies have successfully detected the protein in K562 and Jurkat cell lysates, which can serve as positive controls for troubleshooting experiments .

How should researchers interpret conflicting results between mRNA and protein expression data for OR5AU1?

Discrepancies between OR5AU1 mRNA and protein expression are not uncommon and require careful interpretation:

• Biological explanations:

  • Post-transcriptional regulation may affect translation efficiency

  • Protein stability differences across tissues could lead to accumulation despite similar mRNA levels

  • Tissue-specific alternative splicing might affect antibody recognition sites

• Technical considerations:

  • Antibody specificity issues may result in false positives or negatives

  • Different sensitivities of detection methods (qRT-PCR vs. Western blot/IHC)

  • Sample processing differences between RNA and protein extraction

• Methodological approach to resolve discrepancies:

  • Verify findings with multiple antibodies targeting different epitopes

  • Use multiple detection methods (WB, IHC, IF) for protein validation

  • Perform absolute quantification of mRNA and protein when possible

  • Consider reporter assays (e.g., OR5AU1 promoter driving fluorescent protein) to verify expression patterns

When interpreting results, researchers should note that similar scenarios have been observed with other olfactory receptors, such as OR51J1, where quantitative expression patterns between mRNA and protein don't always align perfectly across tissue types .

What are the key differences between polyclonal and monoclonal antibodies for OR5AU1 research?

When selecting antibodies for OR5AU1 research, understanding the differences between polyclonal and monoclonal options is crucial:

Most commercially available OR5AU1 antibodies mentioned in the search results are rabbit polyclonal antibodies, typically generated using synthetic peptides corresponding to specific regions of the human OR5AU1 protein (e.g., amino acids 201-250) . These polyclonal antibodies have been validated for applications such as Western blot and ELISA, with recommended dilutions ranging from 1:500-1:2000 for Western blot and 1:5000 for ELISA .

How might OR5AU1 antibodies be used in studying olfactory signal transduction mechanisms?

OR5AU1 antibodies offer valuable tools for investigating olfactory signal transduction:

• Receptor localization studies:

  • Immunofluorescence microscopy to visualize OR5AU1 distribution in olfactory epithelium

  • Co-localization with other components of the signaling pathway (Golf, adenylyl cyclase III, CNG channels)

  • Super-resolution microscopy to examine receptor clustering and organization

• Signaling complex isolation:

  • Immunoprecipitation to isolate OR5AU1 and associated proteins

  • Mass spectrometry identification of interacting partners

  • Analysis of post-translational modifications affecting signaling

• Functional correlation:

  • Combine antibody-based detection with calcium imaging or electrophysiology

  • Correlate receptor expression levels with functional responses to odorants

  • Investigate adaptation mechanisms by tracking receptor internalization

• Development and regeneration studies:

  • Track OR5AU1 expression during olfactory neuron development

  • Monitor receptor expression following injury or in aging models

  • Correlate with olfactory function tests

Since OR5AU1 is a member of the G-protein-coupled receptor family arising from single coding-exon genes , antibodies can help elucidate its specific role within the complex landscape of olfactory signal transduction.

What potential exists for OR5AU1 antibodies in diagnostic or biomarker applications?

While primarily research tools, OR5AU1 antibodies might have diagnostic potential based on emerging research on olfactory receptors:

• Cancer biomarker exploration:

  • Research on the related olfactory receptor OR51J1 revealed potential as a cancer-associated biomarker for breast cancer diagnosis

  • OR51J1 showed 2.91-fold higher expression in tumors than normal tissues

  • Similar investigations could explore OR5AU1 expression across cancer types

• Neurological conditions:

  • Olfactory dysfunction occurs in neurodegenerative diseases (Alzheimer's, Parkinson's)

  • Changes in OR5AU1 expression might correlate with disease progression

  • Antibody-based detection could help characterize molecular changes

• Methodological approaches for biomarker validation:

  • Tissue microarray screening across multiple disease states

  • Correlation with clinical outcomes and disease parameters

  • Multivariate analysis combining OR5AU1 with other markers

• Genetic disease correlation:

  • The search results indicated a statistical association between OR5AU1 variants and retinal dystrophy

  • Antibodies could help investigate the biological significance of this genetic association

While diagnostic applications would require extensive validation beyond current research use, the experience with OR51J1 suggests potential for olfactory receptors as disease biomarkers .

How can computational approaches enhance OR5AU1 antibody design and application?

Recent advances in computational biology offer opportunities to improve OR5AU1 antibody research:

• Epitope prediction and antibody design:

  • Computational models can identify optimal epitopes unique to OR5AU1

  • Machine learning approaches can predict cross-reactivity with related olfactory receptors

  • Molecular dynamics simulations can assess epitope accessibility in the native protein

• Specificity enhancement:

  • As demonstrated in research on antibody specificity, computational approaches can design antibodies with customized specificity profiles

  • These models can disentangle binding modes associated with different ligands

  • Such approaches enable the design of antibodies with either specific high affinity for OR5AU1 or controlled cross-specificity

• Structure-based optimization:

  • Homology modeling of OR5AU1 based on related GPCR structures

  • Virtual screening of antibody binding to different receptor conformations

  • In silico mutagenesis to identify stabilizing modifications

• Validation and experimental design:

  • Prediction of optimal experimental conditions for different applications

  • Statistical models to interpret complex expression data

  • Computational tools to assess antibody batch consistency