OR10AD1 Antibody

Which applications are OR10AD1 antibodies validated for?

Based on current validation data, commercially available OR10AD1 antibodies have been successfully used in the following applications:

It's advisable to optimize dilutions for your specific experimental conditions, as these recommendations provide only starting points.

What tissue distribution pattern has been observed for OR10AD1 protein?

OR10AD1 protein has been detected in several tissues, with notable expression in retinal tissue. In human retina specifically, OR10AD1 has been localized to the ciliary region of photoreceptors and in the nuclear envelope of cells in both outer and inner nuclear layers, as well as in ganglion cells . The protein shows a distinctive distribution pattern with predominant localization in the nuclear envelope and, to a lesser extent, in spots within the nucleus . This unusual distribution for a G-protein coupled receptor suggests potential non-canonical functions beyond olfactory sensing.

How can I verify the specificity of OR10AD1 antibody signals in my experiments?

Verification of antibody specificity is crucial for reliable results. Implement the following methodological approaches:

• Peptide competition assay: Pre-incubate the OR10AD1 antibody with the immunizing peptide before application to your sample. This should abolish or significantly reduce specific signals .

• Multiple antibody approach: Use antibodies targeting different epitopes of OR10AD1. Consistent localization patterns across antibodies increase confidence in specificity.

• Genetic controls: If possible, use OR10AD1 knockout or knockdown samples as negative controls, or OR10AD1-overexpressing systems as positive controls.

• Western blot analysis: Confirm single band detection at the expected molecular weight (~72 kDa) despite the calculated weight being ~35.7 kDa, as mentioned earlier .

• Cross-validation: Confirm protein expression correlates with mRNA expression data in your experimental system.

As demonstrated in multiple studies, a properly validated IF experiment with OR10AD1 antibody should show signal reduction or elimination when blocked with the synthesized peptide, confirming specificity .

What are the optimal fixation and permeabilization conditions for OR10AD1 immunostaining?

The subcellular localization of OR10AD1 to both membrane structures (nuclear envelope) and potential nuclear spots requires careful optimization of fixation and permeabilization conditions:

When studying OR10AD1 in photoreceptor cells, consider using centrin-3 as a co-marker for the connecting cilium and basal body complex to properly identify subcellular compartments .

How does the choice of epitope region affect OR10AD1 antibody performance?

Different commercially available OR10AD1 antibodies target distinct epitope regions, which can significantly impact experimental outcomes:

• C-terminal targeting antibodies: These antibodies (like ABIN6258980) recognize the intracellular C-terminal domain . They typically perform well in applications where the protein maintains its native conformation or in denatured conditions where the C-terminus remains accessible.

• Internal region antibodies: Antibodies targeting amino acids 141-190 (like Boster's A30852) recognize an internal sequence . These may perform differently depending on protein folding and membrane topology.

For nuclear envelope localization studies, C-terminal targeting antibodies may provide superior results since this domain is likely more accessible in the nuclear membrane orientation. For studies of potential splice variants or processed forms of OR10AD1, consider using antibodies targeting different regions to obtain complementary data.

What are the recommended controls when studying OR10AD1 in non-olfactory tissues?

OR10AD1 was initially classified as an olfactory receptor but has been detected in non-olfactory tissues such as the retina . When studying OR10AD1 in non-canonical locations, implement these essential controls:

• Tissue-specific positive control: Include a tissue known to express OR10AD1 (e.g., olfactory epithelium) as a positive control.

• mRNA verification: Confirm OR10AD1 transcript expression in your tissue of interest using RT-PCR or RNA-Seq data before antibody-based detection.

• Secondary antibody-only control: To rule out non-specific binding of secondary antibodies.

• Isotype control: Use a non-specific IgG from the same host species as the OR10AD1 antibody to identify background signals.

• Subcellular marker co-localization: When studying retinal tissue, use established markers like centrin-3 (for connecting cilium), β-catenin (for cell adhesion complexes), GM130 (for Cis-Golgi), or LAMP1 (for lysosomes) to verify compartmentalization .

This comprehensive control strategy ensures that the observed signals truly represent OR10AD1 expression rather than technical artifacts.

How can I troubleshoot weak or absent OR10AD1 immunofluorescence signals?

If experiencing challenges with OR10AD1 detection by immunofluorescence, consider this systematic troubleshooting approach:

• Antibody titration: Test a range of antibody concentrations beyond the recommended dilutions (e.g., 1:100 - 1:2000).

• Epitope retrieval optimization: For fixed tissues, particularly formalin-fixed specimens, try heat-induced epitope retrieval methods with citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0).

• Signal amplification: Consider using tyramide signal amplification or a more sensitive detection system.

• Alternative fixation: Compare results between paraformaldehyde fixation and alcohol-based fixatives, as membrane proteins often show differential preservation.

• Detergent adjustment: For nuclear envelope staining, careful optimization of detergent concentration is critical – too much may extract the protein, too little may prevent antibody access.

• Cold methanol step: A brief (-20°C) methanol treatment after fixation can improve access to some epitopes, particularly in membrane structures.

The nuclear envelope localization of OR10AD1 may require specific conditions for optimal visualization, particularly when examining the distribution between the nuclear envelope and intranuclear spots .

How should researchers interpret the discrepancy between OR10AD1's predicted function as an olfactory receptor and its nuclear envelope localization?

The unexpected localization of OR10AD1 to the nuclear envelope represents an intriguing research question . When interpreting such non-canonical findings:

• Consider potential moonlighting functions: GPCRs, including olfactory receptors, can have additional roles beyond their canonical signaling. OR10AD1 may participate in nuclear signaling or gene regulation.

• Evaluate trafficking hypotheses: The nuclear envelope localization could represent a non-functional storage pool, a site of biosynthesis, or a degradation intermediate.

• Examine developmental context: Expression patterns may change during development, with nuclear localization potentially representing a specific developmental state.

• Cross-reference with other ectopically expressed ORs: Compare with other olfactory receptors found outside the olfactory epithelium to identify patterns.

• Investigate potential interactions: Consider nuclear envelope proteins (e.g., lamins, nuclear pore complex components) as potential interaction partners.

This unexpected localization suggests novel functions for OR10AD1 beyond olfactory detection and represents an exciting avenue for future research .

What is the significance of the observed molecular weight difference between predicted and observed OR10AD1 protein size?

The observed molecular weight of approximately 72 kDa significantly exceeds the calculated 35.7 kDa . This discrepancy warrants careful consideration:

• Post-translational modifications: GPCRs often undergo extensive glycosylation, palmitoylation, and other modifications that increase apparent molecular weight.

• Protein-detergent interactions: Incomplete denaturation or detergent binding can alter migration patterns.

• Dimerization: Incomplete reduction of dimers could explain higher molecular weight bands.

• Splice variants: Alternative splicing might produce larger protein isoforms.

To address this discrepancy methodologically:

• Deglycosylation experiments: Treat samples with PNGase F or Endo H before Western blotting to assess contribution of N-linked glycans.

• Reduction optimization: Test stronger reducing conditions to ensure complete monomerization.

• Mass spectrometry: For definitive identification and characterization of the 72 kDa band.

This molecular weight difference is consistent with typical observations for membrane proteins, particularly GPCRs, and should not be interpreted as non-specific binding without further evidence.

What is the evidence for cross-reactivity of human OR10AD1 antibodies with other species?

Available OR10AD1 antibodies show different cross-reactivity profiles:

For feline tissues specifically, while not explicitly validated, there is a reasonable possibility of cross-reactivity with some antibodies. A researcher inquiry noted in the search results asked about using the Boster antibody (A30852) on feline tissues, and the manufacturer suggested there is "a good chance of cross-reactivity" despite lack of validation .

When planning to use these antibodies in non-validated species, preliminary testing with positive controls is essential, and sequence alignment analysis between human OR10AD1 and the target species' ortholog can help predict potential cross-reactivity.

What methodological considerations apply when studying OR10AD1 in retinal tissue?

The detection of OR10AD1 in human retina requires specialized approaches :

• Tissue preservation: Fresh retinal tissue should be processed rapidly, with careful cryoprotection to maintain delicate structures like the photoreceptor outer segments.

• Orientation and sectioning: Proper orientation of retinal samples is critical for interpreting layered structure. Use vertical sections to visualize all retinal layers.

• Co-labeling strategy: For precise localization, co-stain with:

  • Centrin-3 to identify connecting cilium and basal body

  • Nuclear markers (DAPI) to confirm nuclear envelope localization

  • Layer-specific markers to identify retinal layers (ganglion cell, inner nuclear, outer nuclear)

• Confocal microscopy optimization: Use z-stacking with thin optical sections (0.3-0.5 μm) to distinguish nuclear envelope from intranuclear localization.

• 3D reconstruction: Consider 3D reconstruction of confocal z-stacks to fully characterize the distribution pattern of OR10AD1 in nuclear structures.

These approaches have successfully demonstrated that OR10AD1 localizes to the ciliary region of photoreceptors and the nuclear envelope of several retinal cell types, suggesting potential roles in nuclear transport or signaling in retinal neurons .