OR2S2 Antibody

What is OR2S2 and what is its role in human physiology?

OR2S2 (Olfactory Receptor Family 2 Subfamily S Member 2) is a protein-coding gene that belongs to the large family of olfactory receptors. These receptors interact with odorant molecules in the nose to initiate neuronal responses that trigger the perception of smell. OR2S2 is a G-protein-coupled receptor (GPCR) with a 7-transmembrane domain structure, similar to many neurotransmitter and hormone receptors .

Physiologically, OR2S2 contributes to the olfactory system's ability to detect and distinguish between different odors. The protein is part of the largest gene family in the human genome - the olfactory receptor family - and plays a specific role in the recognition and G protein-mediated transduction of odorant signals .

What is the genomic context and structure of the OR2S2 gene?

The OR2S2 gene is located on the short arm of chromosome 9 at position 9p13.3. Its genomic sequence is found on chromosome 9 (NC_000009.12) at positions 35957108 to 35958154 on the complement strand. A notable characteristic of the OR2S2 gene is that it has only one exon, which is typical for olfactory receptor genes .

The calculated molecular weight of the OR2S2 protein is approximately 35-40 kDa, with some antibody suppliers reporting an observed molecular weight of around 35 kDa .

What applications are OR2S2 antibodies typically used for in research?

OR2S2 antibodies are used in multiple research applications including:

These applications allow researchers to study OR2S2 expression, localization, and function in various experimental contexts .

What is the significance of OR2S2 being classified as a segregating pseudogene?

OR2S2 is described as a segregating pseudogene, meaning that genetic variation exists within the human population regarding this gene's functionality. Some individuals possess an allele that encodes a functional olfactory receptor, while others have an allele encoding a protein predicted to be non-functional .

This characteristic makes OR2S2 particularly interesting for research into olfactory receptor evolution, genetic diversity, and potentially individual differences in olfactory perception. The segregating nature of this gene may contribute to variability in smell perception among different people and could be relevant to studies of olfactory system development and function .

How should researchers validate OR2S2 antibody specificity for experimental use?

Antibody validation is critical for OR2S2 research due to the high sequence similarity among olfactory receptors. A comprehensive validation approach should include:

• Western blot analysis with positive and negative controls: Use tissues/cells known to express OR2S2 as positive controls, and knockdown/knockout samples as negative controls. Look for a single band at the expected molecular weight (~35 kDa) .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm signal elimination. Most OR2S2 antibodies are raised against synthetic peptide immunogens, such as the peptide sequence from human OR2S2 protein positions 262-276 AA .

• Cross-species reactivity testing: Validate the antibody in all species of interest. For example, some commercially available OR2S2 antibodies react with human, mouse, and rat samples, which should be independently verified .

• Multiple detection methods: Compare results across different techniques (WB, IHC, IF) to ensure consistent detection patterns.

• Knockdown verification: Use siRNA or shRNA against OR2S2 to confirm signal reduction with the antibody.

What are the optimal protocols for using OR2S2 antibodies in Western blot applications?

For optimal Western blot detection of OR2S2, the following methodology is recommended:

• Sample preparation:

  • Extract proteins from tissues or cells using appropriate lysis buffers containing protease inhibitors

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

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal separation around the 35 kDa mark

  • Transfer to PVDF or nitrocellulose membranes at 100V for 60-90 minutes

• Blocking and antibody incubation:

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

  • Incubate with primary OR2S2 antibody at dilutions of 1:500-1:2000 overnight at 4°C

  • Use appropriate HRP-conjugated secondary antibodies at 1:5000-1:10000 dilution

• Detection and troubleshooting:

  • Use enhanced chemiluminescence detection systems

  • Expected molecular weight: ~35 kDa

  • For weak signals, extend primary antibody incubation time or increase concentration

  • For high background, increase washing steps or adjust blocking conditions

• Controls:

  • Include positive control tissue known to express OR2S2

  • Use appropriate loading controls (β-actin, GAPDH) to normalize expression levels

What are the methodological considerations for immunohistochemical detection of OR2S2?

Successful immunohistochemical detection of OR2S2 requires careful attention to:

• Tissue preparation and fixation:

  • Fix tissues in 4% paraformaldehyde for 24-48 hours

  • Paraffin embedding should be performed with careful temperature control

  • Section thickness of 4-6 μm is optimal for OR2S2 detection

• Antigen retrieval:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) is generally effective

  • Pressure cooking for 3 minutes or microwave treatment for 10-15 minutes

  • Allow sections to cool slowly to room temperature

• Antibody dilution and incubation:

  • Use OR2S2 antibodies at 1:50-1:200 dilution

  • Incubate overnight at 4°C in a humidified chamber

  • Use appropriate detection systems (HRP/DAB or fluorescent)

• Controls and counterstaining:

  • Include positive control tissues (nasal epithelium)

  • Use isotype controls to assess non-specific binding

  • Mild hematoxylin counterstain for better tissue visualization

• Signal optimization:

  • Titrate antibody concentration for optimal signal-to-noise ratio

  • Consider signal amplification systems for low expression levels

How can researchers address potential cross-reactivity with other olfactory receptors?

The high sequence homology among olfactory receptors presents a significant challenge. To address potential cross-reactivity:

• Antibody selection:

  • Choose antibodies raised against unique epitopes of OR2S2

  • Polyclonal antibodies like those available commercially are generated against specific peptide sequences (e.g., positions 262-276 AA or 241-290)

• Preabsorption controls:

  • Perform preabsorption with the immunizing peptide

  • Consider preabsorption with peptides from closely related receptors to test specificity

• Complementary approaches:

  • Validate antibody results with mRNA detection methods (in situ hybridization or RT-PCR)

  • Use multiple antibodies targeting different epitopes of OR2S2

• Knockout/knockdown validation:

  • Use CRISPR/Cas9 or siRNA approaches to create negative controls

  • Compare staining patterns in tissues with known differential expression

• Bioinformatic analysis:

  • Conduct sequence alignment of the immunizing peptide against the proteome

  • Identify potentially cross-reactive proteins in silico

What experimental approaches can be used to study OR2S2 function in olfactory signaling?

To investigate OR2S2 function in olfactory signaling, researchers can employ several complementary approaches:

• Heterologous expression systems:

  • Express OR2S2 in HEK293 or other cell lines

  • Conduct calcium imaging or cAMP assays to identify ligands

  • Co-express with olfactory G proteins (Golf) for functional coupling

• Primary culture studies:

  • Isolate and culture olfactory sensory neurons

  • Use OR2S2 antibodies to identify OR2S2-expressing neurons

  • Perform electrophysiological recordings to measure responses to potential ligands

• In vivo approaches:

  • Generate reporter mice with fluorescent proteins driven by the OR2S2 promoter

  • Conduct in vivo calcium imaging of olfactory bulb responses

  • Perform behavioral assays to assess olfactory discrimination

• Molecular interaction studies:

  • Use co-immunoprecipitation with OR2S2 antibodies to identify interacting proteins

  • Employ proximity ligation assays to visualize protein interactions in situ

  • Utilize FRET/BRET approaches to study receptor dynamics

• Genetic studies:

  • Analyze OR2S2 polymorphisms and their correlation with olfactory perception

  • Investigate the functional consequences of the segregating pseudogene nature of OR2S2

How can researchers investigate the significance of OR2S2 being a segregating pseudogene?

The segregating pseudogene status of OR2S2 presents unique research opportunities:

• Population genetics approaches:

  • Sequence OR2S2 across diverse human populations

  • Analyze the frequency of functional vs. non-functional alleles

  • Correlate genotypes with olfactory phenotypes

• Functional characterization:

  • Express both functional and non-functional alleles in heterologous systems

  • Compare receptor properties including trafficking, ligand binding, and signaling

  • Use site-directed mutagenesis to identify critical residues

• Transcriptional analysis:

  • Quantify expression levels of functional vs. non-functional alleles

  • Investigate potential nonsense-mediated decay of transcripts from non-functional alleles

  • Compare expression patterns across tissues

• Evolutionary studies:

  • Conduct comparative genomics of OR2S2 across species

  • Calculate selection pressures on functional vs. non-functional variants

  • Trace the evolutionary history of OR2S2 pseudogenization

• Clinical correlations:

  • Investigate associations between OR2S2 allele status and olfactory function

  • Explore potential links to conditions with altered olfactory perception

What control experiments are essential when using OR2S2 antibodies?

Robust experimental design requires appropriate controls when using OR2S2 antibodies:

Implementing these controls ensures reliable and interpretable results when studying OR2S2 expression and function.

How can OR2S2 antibodies be combined with other techniques for comprehensive olfactory research?

Integrating OR2S2 antibody techniques with complementary methods enhances research depth:

• Combined immunohistochemistry and in situ hybridization:

  • Co-localize OR2S2 protein and mRNA expression

  • Assess correlation between transcription and translation

  • Identify cells expressing functional vs. non-functional alleles

• Multi-label immunofluorescence:

  • Combine OR2S2 antibodies with markers for olfactory neuron subtypes

  • Co-stain with signaling pathway components (Golf, ACIII)

  • Investigate co-expression with other olfactory receptors

• Tissue clearing and 3D imaging:

  • Apply OR2S2 antibodies to cleared tissue preparations

  • Perform whole-mount immunostaining of olfactory epithelium

  • Create 3D reconstructions of OR2S2-expressing neuron distributions

• Single-cell approaches:

  • Combine immunostaining with laser capture microdissection

  • Correlate OR2S2 protein expression with single-cell transcriptomics

  • Perform patch-clamp electrophysiology on identified OR2S2-expressing neurons

• Proximity labeling techniques:

  • Fuse OR2S2 with BioID or APEX2 for proximity proteomics

  • Identify the OR2S2 interactome in native contexts

  • Map the molecular organization of OR2S2 signaling complexes

What emerging technologies might enhance OR2S2 research beyond traditional antibody-based methods?

Several cutting-edge approaches show promise for advancing OR2S2 research:

• CRISPR-based technologies:

  • Generate OR2S2 knockout models to study function

  • Create knock-in reporter lines to visualize expression

  • Employ CRISPRa/CRISPRi for controlled expression modulation

• Super-resolution microscopy:

  • Apply STORM, PALM, or STED microscopy with OR2S2 antibodies

  • Resolve nanoscale organization of OR2S2 in olfactory cilia

  • Investigate receptor clustering and trafficking

• Spatial transcriptomics:

  • Correlate OR2S2 protein localization with spatial gene expression patterns

  • Map the molecular context of OR2S2-expressing neurons

  • Identify spatial relationships between different olfactory receptor zones

• Computational approaches:

  • Apply machine learning to analyze OR2S2 expression patterns

  • Use molecular dynamics simulations to study ligand interactions

  • Develop predictive models of OR2S2 functionality based on sequence

• Organoid models:

  • Generate olfactory epithelium organoids expressing OR2S2

  • Study receptor development and function in 3D culture

  • Test responses to odorants in controlled environments

How should researchers interpret contradictory results when using different OR2S2 antibodies?

When facing discrepancies between different OR2S2 antibodies:

• Epitope comparison:

  • Determine the immunizing peptides for each antibody

  • Different antibodies may target distinct regions (e.g., positions 262-276 AA vs. 241-290)

  • Consider potential disruption of epitopes by protein modifications or conformation

• Validation stringency assessment:

  • Review validation data for each antibody

  • Evaluate the comprehensiveness of specificity testing

  • Consider the robustness of positive and negative controls

• Technical variables:

  • Compare fixation and antigen retrieval protocols

  • Assess differences in detection systems

  • Evaluate antibody format (e.g., native vs. HRP-conjugated)

• Integration with non-antibody methods:

  • Correlate results with mRNA expression data

  • Consider functional data from reporter systems

  • Evaluate consistency with expected expression patterns

• Experimental design to resolve discrepancies:

  • Perform side-by-side comparisons under identical conditions

  • Use multiple antibodies in the same experiment when possible

  • Consider epitope masking or accessibility in different contexts

Understanding the basis for contradictory results can provide valuable insights into OR2S2 biology and improve experimental design for future studies.

What are common challenges when using OR2S2 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with OR2S2 antibodies:

Methodical troubleshooting and optimization can significantly improve results when working with OR2S2 antibodies.

How can researchers optimize fixation and antigen retrieval for OR2S2 detection in tissue sections?

Optimizing fixation and antigen retrieval is critical for successful OR2S2 detection:

• Fixation optimization:

  • Compare 4% paraformaldehyde, 10% neutral buffered formalin, and Bouin's fixative

  • Test fixation times from 4-48 hours to balance preservation and epitope accessibility

  • Consider brief post-fixation in cold methanol to improve membrane protein detection

• Antigen retrieval methods comparison:

  • Heat-induced epitope retrieval (HIER):

    • Citrate buffer (pH 6.0)

    • EDTA buffer (pH 8.0)

    • Tris-EDTA buffer (pH 9.0)

  • Enzymatic retrieval:

    • Proteinase K (1-5 μg/ml for 5-15 minutes)

    • Trypsin (0.05% for 10-20 minutes)

• Retrieval conditions optimization:

  • Test different heating methods (microwave, pressure cooker, water bath)

  • Vary treatment duration (10-30 minutes)

  • Optimize cooling conditions (rapid vs. slow cooling)

• Tissue-specific considerations:

  • Nasal epithelium may require gentler conditions due to fragility

  • Consider section thickness (thinner sections may require milder retrieval)

  • Adjust protocols based on tissue processing history

• Sequential antigen retrieval:

  • Apply mild HIER followed by brief enzymatic treatment

  • Use stepwise heating with gradual temperature increases

  • Consider dual buffer systems for comprehensive epitope exposure

These optimization steps can significantly improve the detection of OR2S2 in tissue sections, particularly in challenging samples like olfactory epithelium.