OR10R2 Antibody

What is OR10R2 and what is its functional significance?

OR10R2 is a member of the olfactory receptor family, specifically belonging to subfamily 10R member 2. It is a G-protein coupled receptor with a calculated molecular weight of approximately 36-37 kDa . This receptor is classified as a multi-pass membrane protein primarily located within the cell membrane . While traditionally associated with olfactory function, researchers should be aware that its expression patterns and potential roles in non-olfactory tissues remain an active area of investigation.

The protein is encoded by the OR10R2 gene, which interestingly overlaps with a thymus-specific noncoding RNA (Thy-ncR1) on the antisense strand . This genomic arrangement raises intriguing questions about potential regulatory relationships and tissue-specific expression patterns that may be relevant to experimental design.

What applications are OR10R2 antibodies validated for?

Based on current literature and commercial availability, OR10R2 antibodies have been validated for several common immunological techniques:

When planning experiments, it's advisable to perform preliminary validation in your specific experimental system, as antibody performance can vary significantly between applications and sample preparations .

What are the key specifications of commercially available OR10R2 antibodies?

Multiple suppliers offer polyclonal antibodies against OR10R2, with rabbit being the predominant host species for immunization . These antibodies are typically generated using synthetic peptides derived from human OR10R2 sequences. One specific antibody was developed using a peptide corresponding to amino acids 271-320 of the human OR10R2 protein .

For research applications, it's important to note that these antibodies primarily demonstrate reactivity against human OR10R2, though some may cross-react with orthologous proteins from other species based on sequence conservation .

How is OR10R2 gene expression regulated in different tissues?

Intriguingly, the OR10R2 gene occupies the antisense strand of the Thy-ncR1 gene, a thymus-specific noncoding RNA located at 1q23.1 on human chromosome 1 . This genomic arrangement raises questions about potential regulatory interactions between these overlapping genes.

Research has shown that while Thy-ncR1 is highly expressed in thymus tissue and Jurkat T-cell leukemia cells, the OR10R2 gene appears to be undetectable in these same tissues . This observation suggests tissue-specific regulation mechanisms that control OR10R2 expression, with expression traditionally expected to be restricted to the olfactory bulb. When designing experiments targeting OR10R2, researchers should carefully consider these tissue-specific expression patterns and potential regulatory mechanisms.

What validation strategies should be employed for OR10R2 antibodies?

Antibody validation is critical for ensuring experimental reproducibility. For OR10R2 antibodies, consider implementing these validation strategies:

• Positive and negative controls: Use cell lines or tissues with known OR10R2 expression levels. HT-29 cells have been documented as a positive control in western blot applications .

• Peptide competition assay: Pre-incubate the antibody with its immunizing peptide to confirm specificity of detection.

• Knockout/knockdown validation: Although challenging for olfactory receptors, CRISPR/Cas9 knockout or siRNA knockdown can provide definitive validation.

• Cross-application verification: Confirm target detection using multiple techniques (e.g., WB, IF/ICC) to establish consistent detection patterns.

• Molecular weight verification: Confirm that the detected protein band corresponds to the predicted molecular weight of OR10R2 (approximately 36-37 kDa) .

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

Due to sequence homology among olfactory receptor family members, cross-reactivity is a significant concern. Consider these approaches:

• Epitope analysis: Compare the immunogen sequence used to generate the antibody against other OR family members using sequence alignment tools.

• Multiple antibody approach: Utilize antibodies targeting different epitopes of OR10R2 to confirm specificity.

• Mass spectrometry validation: For definitive identification, consider immunoprecipitation followed by mass spectrometry to confirm the identity of the detected protein.

• Species prediction scoring: Review available cross-species reactivity prediction data. For example, some OR10R2 antibodies show potential cross-reactivity with bovine and rat orthologs, but with varying confidence scores .

What are the implications of OR10R2's relationship with Thy-ncR1 for experimental design?

The overlapping genomic arrangement of OR10R2 and Thy-ncR1 presents unique experimental considerations:

• Antisense regulation: When studying OR10R2 expression, consider the potential regulatory influence of Thy-ncR1. RNase protection assays have been used to simultaneously assess expression of both transcripts .

• Tissue context: While OR10R2 expression appears absent in thymus and Jurkat cells despite high Thy-ncR1 expression, other tissue contexts might reveal different regulatory relationships .

• Transcriptional interference: Design primers and probes carefully to distinguish between sense and antisense transcripts when performing RT-PCR or RNA hybridization experiments.

• Functional studies: Consider that Thy-ncR1 has "T-cell-specific function(s) independent of OR gene expression," which may influence experimental interpretation in immune cell contexts .

What are the critical parameters for optimizing western blot detection of OR10R2?

Western blot optimization for membrane proteins like OR10R2 requires careful consideration:

• Sample preparation: Use specialized lysis buffers containing detergents suitable for membrane protein solubilization (e.g., RIPA buffer with 0.1% SDS or dedicated membrane protein extraction kits).

• Protein denaturation: Avoid excessive heating which can cause aggregation of membrane proteins; 37°C for 30 minutes may be preferable to boiling.

• Gel percentage selection: Use 10-12% acrylamide gels for optimal resolution around 36-37 kDa.

• Transfer conditions: Implement wet transfer methods with methanol-containing buffers to facilitate transfer of hydrophobic membrane proteins.

• Blocking optimization: Test both BSA and milk-based blocking solutions, as membrane proteins sometimes demonstrate different background patterns with each.

• Antibody dilution: Start with the recommended dilution range (1:500-1:1000) and optimize based on signal-to-noise ratio .

• Detection system selection: Enhanced chemiluminescence (ECL) systems are commonly used, but fluorescent secondary antibodies may provide better quantitative results.

How can researchers leverage OR10R2 antibodies in multi-parameter analyses?

For comprehensive characterization of OR10R2 in complex biological systems:

• Multiplex immunofluorescence: Combine OR10R2 antibodies with markers for subcellular compartments or interacting proteins to assess co-localization.

• Flow cytometry applications: Although not explicitly validated in the literature, polyclonal antibodies against cell surface proteins can potentially be adapted for flow cytometry following careful optimization.

• Proximity ligation assays: Investigate protein-protein interactions involving OR10R2 by combining antibodies against potential interacting partners.

• ChIP-sequencing approaches: For regulatory studies investigating the genomic context of OR10R2 and Thy-ncR1, chromatin immunoprecipitation using appropriate antibodies (e.g., against transcription factors or histone modifications) may provide valuable insights.

What controls should be included when using OR10R2 antibodies?

Proper experimental controls are essential for interpretable results:

• Primary antibody controls: Include samples processed without primary antibody to assess secondary antibody specificity.

• Isotype controls: Include matched isotype controls (rabbit IgG for polyclonal rabbit antibodies) to assess non-specific binding.

• Tissue/cell type controls: Include samples known to express OR10R2 (e.g., olfactory tissues) and samples expected to lack expression (e.g., thymus tissue) .

• Loading controls: For western blot applications, include appropriate loading controls specific to the subcellular fraction being analyzed (e.g., Na⁺/K⁺ ATPase for membrane proteins).

• Peptide competition: Pre-incubate antibody with immunizing peptide to demonstrate signal specificity.

What are best practices for immunohistochemical detection of OR10R2?

When performing IHC with OR10R2 antibodies:

• Antigen retrieval optimization: Test multiple antigen retrieval methods, as membrane proteins often require specific conditions for epitope exposure.

• Fixation considerations: Compare formaldehyde-fixed and fresh-frozen samples, as some epitopes may be sensitive to cross-linking fixatives.

• Signal amplification: Consider tyramide signal amplification or other enhancement methods if signal strength is limited.

• Counterstaining selection: Choose counterstains that will not interfere with visualization of the expected subcellular localization (membrane).

• Positive control tissues: Include olfactory epithelium sections as positive controls when possible.

• Negative control tissues: Based on available literature, thymus tissue appears to lack OR10R2 expression and could serve as a negative control .

How can OR10R2 antibodies contribute to understanding ectopic expression of olfactory receptors?

While traditionally viewed as olfactory bulb-specific, emerging research suggests olfactory receptors may have extra-nasal functions:

• Tissue screening approaches: Systematic screening of diverse tissue types using validated OR10R2 antibodies could reveal unexpected expression patterns.

• Single-cell analysis: Combining OR10R2 antibodies with single-cell techniques could identify rare cell populations expressing this receptor outside the olfactory system.

• Functional implications: Investigation of OR10R2 in non-olfactory contexts could reveal novel signaling pathways and biological functions.

• Disease associations: Exploring potential dysregulation of OR10R2 expression in disease states might uncover pathological significance.

What advances in antibody technology might enhance OR10R2 research?

The field of antibody development continues to evolve:

• Recombinant antibody approaches: Development of recombinant monoclonal antibodies against OR10R2 could improve reproducibility compared to polyclonal reagents.

• Nanobody development: Single-domain antibodies derived from camelids might provide access to epitopes challenging for conventional antibodies.

• Machine learning for antibody design: Recent advances in iterative machine learning-based design of biological tools, as demonstrated in enhancer design , could potentially be applied to develop more specific antibodies against challenging targets like OR10R2.

• Application-specific antibody engineering: Modification of existing antibodies with application-specific tags (e.g., cell-penetrating peptides, fluorophores) could expand their utility.