OR10K1/OR10K2 Antibody

What are OR10K1/OR10K2 proteins and what is their biological significance?

OR10K1 and OR10K2 are members of the olfactory receptor family, specifically belonging to subfamily 10. These G-protein coupled receptors are primarily involved in olfactory signal transduction. While traditionally associated with sensory functions, recent research has identified differential expression patterns of these receptors in various tissues beyond the olfactory epithelium, suggesting potential roles in other physiological processes. Notably, OR10K1 has been identified among the top downregulated differentially expressed genes (DEGs) in certain immune microenvironment studies, while OR10K2 appears among upregulated genes in similar contexts, indicating potentially divergent biological functions .

What is the expected molecular weight of OR10K1/OR10K2 proteins in Western blot applications?

The observed molecular weight for OR10K1/OR10K2 in Western blot applications is approximately 72 kDa, though the calculated molecular weight based on amino acid sequence is approximately 35 kDa (35,079 Da specifically) . This discrepancy is not uncommon for membrane proteins like olfactory receptors, which often demonstrate higher apparent molecular weights on SDS-PAGE due to post-translational modifications (particularly glycosylation) and the hydrophobic nature of transmembrane domains affecting SDS binding and protein migration patterns. When troubleshooting, researchers should anticipate this higher molecular weight band rather than the theoretical weight calculated from the primary sequence .

What tissue/cell specificity should researchers expect when working with OR10K1/OR10K2 antibodies?

Based on available antibody specifications, OR10K1/OR10K2 antibodies primarily demonstrate reactivity with human, mouse, and rat samples . While traditionally associated with olfactory epithelium, researchers should be aware that olfactory receptors have been detected in multiple non-olfactory tissues. Recent research has implicated these receptors in immune microenvironment contexts, suggesting expression in additional tissues beyond primary sensory organs . When designing experiments, researchers should include appropriate positive control tissues, particularly when investigating novel expression patterns in non-canonical locations.

What critical validation steps should researchers perform before using OR10K1/OR10K2 antibodies in their experiments?

Validation of OR10K1/OR10K2 antibodies should follow a multi-step approach:

• Western blot validation: Confirm detection of a band at the expected 72 kDa molecular weight in appropriate positive control samples .

• Knockout/knockdown validation: If possible, compare signal between wild-type samples and those where OR10K1/OR10K2 expression has been genetically reduced.

• Peptide competition assay: Pre-incubate antibody with the immunogen peptide (derived from the internal region of human OR10K1/2) to confirm signal specificity .

• Cross-reactivity assessment: Test the antibody against recombinant OR10K1 and OR10K2 separately to determine specificity for each target.

• Application-specific validation: For each intended application (WB, ICC/IF, ELISA), perform concentration gradients to determine optimal working dilutions as suggested in the product specifications .

How can researchers distinguish between OR10K1 and OR10K2 signals given their sequence similarities?

Distinguishing between OR10K1 and OR10K2 signals presents a technical challenge due to their sequence homology. Most commercially available antibodies recognize both proteins, as evidenced by product naming conventions (OR10K1/OR10K2) . To differentiate between these targets:

• Complement antibody-based detection with transcript-specific qPCR to determine which isoform predominates in your experimental system.

• Consider using epitope-tagged recombinant constructs for each receptor when overexpression is possible.

• Employ competitive binding assays with peptides specific to non-conserved regions of each receptor.

• Use bioinformatic analysis to identify unique epitopes and potentially develop custom antibodies targeting these regions if commercially available options cannot provide sufficient discrimination.

• Implement orthogonal methods like mass spectrometry for definitive protein identification in complex samples.

What are the optimal storage conditions for maintaining OR10K1/OR10K2 antibody activity?

For optimal preservation of OR10K1/OR10K2 antibody activity, researchers should:

• Store antibody aliquots at -20°C for long-term storage .

• For short-term storage and frequent use (up to one month), store at 4°C to minimize freeze-thaw cycles .

• Prepare small working aliquots upon receipt to avoid repeated freeze-thaw cycles, which significantly diminish antibody performance .

• Store in buffer containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide as specified by manufacturers to maintain stability .

• When retrieving from frozen storage, thaw antibodies gradually on ice rather than at room temperature to preserve binding capacity.

• Monitor antibody performance periodically using positive controls to ensure continued reactivity over the storage period.

What are the optimized protocols for using OR10K1/OR10K2 antibodies in Western blotting applications?

For optimal Western blotting results with OR10K1/OR10K2 antibodies:

• Sample preparation: Extract proteins using RIPA buffer supplemented with protease inhibitors; heat samples at 95°C for 5 minutes in reducing sample buffer.

• Gel selection: Use 10% SDS-PAGE gels for optimal resolution of the 72 kDa target protein .

• Transfer conditions: Transfer to PVDF membrane at 100V for 60-90 minutes in cold transfer buffer containing 10-20% methanol.

• Blocking: Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Primary antibody: Dilute OR10K1/OR10K2 antibody at 1:500 to 1:2000 in blocking buffer and incubate overnight at 4°C .

• Washing: Wash membranes 3-5 times with TBST, 5-10 minutes per wash.

• Secondary antibody: Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour at room temperature.

• Detection: Develop using enhanced chemiluminescence and expect a band at approximately 72 kDa .

• Controls: Include positive control tissues and negative controls (secondary antibody only) in each experiment.

What protocol modifications are recommended for immunofluorescence applications with OR10K1/OR10K2 antibodies?

For immunofluorescence staining using OR10K1/OR10K2 antibodies:

• Fixation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature or tissues with 10% neutral buffered formalin.

• Permeabilization: Permeabilize with 0.1-0.2% Triton X-100 in PBS for 10 minutes for intracellular epitope access.

• Blocking: Block with 5% normal serum (from the species of secondary antibody) in PBS with 0.1% Tween-20 for 1 hour.

• Primary antibody: Dilute OR10K1/OR10K2 antibody at 1:200 to 1:1000 in blocking buffer and incubate overnight at 4°C .

• Washing: Wash 3 times with PBS-T, 5 minutes each.

• Secondary antibody: Incubate with fluorochrome-conjugated anti-rabbit secondary antibody at 1:500 in blocking buffer for 1 hour at room temperature.

• Nuclear counterstaining: Counterstain with DAPI (1:1000) for 5 minutes.

• Mounting: Mount with antifade mounting medium and seal with nail polish.

• Controls: Include peptide competition controls and secondary-only controls to confirm specificity.

• Imaging parameters: Begin imaging with lower exposure settings and adjust as needed, as olfactory receptors often show relatively low expression levels in non-olfactory tissues.

What are the critical considerations for ELISA applications using OR10K1/OR10K2 antibodies?

For ELISA applications using OR10K1/OR10K2 antibodies:

• Antibody dilution: Use a high dilution factor of 1:20,000 for ELISA applications, significantly higher than for other applications like WB (1:500-1:2000) or IF/ICC (1:200-1:1000) .

• Sample types: The antibody has been tested with cell culture supernatant, plasma, serum, and tissue homogenate samples .

• Assay optimization: Perform preliminary titration experiments to determine optimal coating concentration, blocking conditions, and incubation times.

• Controls: Include a standard curve using recombinant OR10K1/OR10K2 protein if available, or use samples with known expression levels as positive controls.

• Cross-reactivity prevention: Pre-absorb the antibody with related proteins if cross-reactivity is a concern, particularly with other olfactory receptor family members.

• Signal development: Monitor colorimetric development carefully, as signal-to-noise ratio can be challenging with low-abundance membrane proteins.

• Data analysis: Use appropriate statistical methods to determine the limit of detection and quantification range for your specific experimental conditions.

• Validation: Confirm ELISA results with orthogonal methods such as Western blotting when establishing the assay for novel sample types.

How can researchers address weak or absent signals when using OR10K1/OR10K2 antibodies?

When facing weak or absent signals with OR10K1/OR10K2 antibodies:

• Sample preparation: Ensure adequate protein extraction, particularly for membrane proteins, by using detergent-containing buffers (RIPA or NP-40) with complete protease inhibitor cocktails.

• Antibody concentration: Increase primary antibody concentration; try the lower end of the recommended dilution range (1:500 for WB, 1:200 for IF/ICC) .

• Incubation conditions: Extend primary antibody incubation to overnight at 4°C to increase binding efficiency.

• Detection system: Switch to a more sensitive detection system (e.g., from colorimetric to chemiluminescent for WB, or implement signal amplification for IF).

• Epitope retrieval: For fixed tissues/cells, optimize antigen retrieval methods (heat-induced or enzymatic) to improve epitope accessibility.

• Expression levels: Verify target expression in your samples via RT-PCR before immunodetection, as OR10K1/OR10K2 may have tissue-specific expression patterns.

• Antibody quality: Check antibody viability by testing on positive control samples known to express the target protein.

• Buffer conditions: Ensure the antibody storage buffer maintains proper pH and contains stabilizers as recommended (50% glycerol, 0.5% BSA, 0.02% sodium azide) .

What strategies can researchers employ when facing high background or non-specific signals?

To reduce background and non-specific signals when working with OR10K1/OR10K2 antibodies:

• Blocking optimization: Extend blocking time (2-3 hours) or switch blocking agents (BSA, normal serum, commercial blockers) to find optimal conditions.

• Antibody dilution: Increase dilution of primary antibody; test the upper end of recommended ranges (1:2000 for WB, 1:1000 for IF/ICC) .

• Washing stringency: Increase the number and duration of washes (5-6 washes of 10 minutes each with 0.1% Tween-20 in PBS/TBS).

• Secondary antibody: Dilute secondary antibody further or pre-absorb with tissue/cell lysate from the species being analyzed.

• Cross-adsorption: Pre-incubate primary antibody with non-specific proteins (e.g., non-relevant tissue lysate) to reduce non-specific binding.

• Detergent adjustment: Increase detergent concentration in washing and antibody dilution buffers (0.2-0.3% Tween-20 or 0.1% Triton X-100).

• Fixation optimization: For IF applications, test different fixation methods as over-fixation can increase background.

• Filter selection: For fluorescence applications, ensure proper filter sets to minimize autofluorescence from tissues.

• Endogenous enzyme blocking: For IHC applications, block endogenous peroxidase or phosphatase activity before antibody incubation.

How should researchers interpret unexpected molecular weight bands when using OR10K1/OR10K2 antibodies?

When encountering unexpected bands with OR10K1/OR10K2 antibodies:

• Expected vs. observed weight: While the calculated molecular weight is approximately 35 kDa, the observed weight is typically 72 kDa due to post-translational modifications and membrane protein properties .

• Higher molecular weight bands: Bands above 72 kDa may represent:

  • Glycosylated forms (olfactory receptors often undergo N-linked glycosylation)

  • Dimers or oligomers if sample preparation was insufficient to fully denature complexes

  • Ubiquitinated forms marking the protein for degradation

• Lower molecular weight bands: Bands below 72 kDa may indicate:

  • Proteolytic degradation products (enhance protease inhibition during sample preparation)

  • Splice variants (verify with transcript analysis)

  • Cross-reactivity with related olfactory receptor family members

• Validation approaches:

  • Peptide competition assay to confirm which bands disappear when the antibody is pre-absorbed with the immunizing peptide

  • Sample treatment with glycosidases to identify glycosylated forms

  • Mass spectrometry analysis of excised bands for definitive identification

• Literature comparison: Compare your results with published Western blot images of OR10K1/OR10K2 to identify common patterns.

How can OR10K1/OR10K2 antibodies be utilized in studying the immune microenvironment of thymic epithelial tumors?

OR10K1/OR10K2 antibodies can be valuable tools in investigating thymic epithelial tumor (THYM) immune microenvironments:

• Expression pattern analysis: Use immunohistochemistry or immunofluorescence with OR10K1/OR10K2 antibodies to map receptor distribution in different THYM immunotypes. This is particularly relevant as OR10K1 has been identified among top downregulated genes in certain THYM immunotypes, while OR10K2 appears among upregulated genes .

• Correlation studies: Combine OR10K1/OR10K2 immunostaining with immune cell markers to correlate receptor expression with immune infiltration patterns.

• Prognostic assessment: Evaluate OR10K1/OR10K2 expression levels as potential prognostic biomarkers in THYM patient cohorts, given their inclusion in an 11-gene prognostic model .

• Functional investigations:

  • Co-immunoprecipitation using validated OR10K1/OR10K2 antibodies to identify binding partners in tumor cells

  • Chromatin immunoprecipitation (ChIP) assays with transcription factors potentially regulating OR10K1/OR10K2 expression

• Therapeutic response monitoring: Assess changes in OR10K1/OR10K2 expression before and after immunotherapeutic interventions.

• Multi-omics integration: Combine antibody-based protein detection with transcriptomic data to build comprehensive models of receptor involvement in tumor immune microenvironments.

What methodological approaches can researchers use to investigate potential functional differences between OR10K1 and OR10K2?

To investigate functional differences between OR10K1 and OR10K2:

• Differential expression analysis:

  • Use antibodies in combination with transcript-specific probes to map differential expression patterns across tissues

  • Quantify relative abundance ratios between OR10K1 and OR10K2 in different physiological and pathological states

• Receptor-specific knockdown/knockout:

  • Design siRNA or CRISPR approaches targeting unique regions of each receptor

  • Use OR10K1/OR10K2 antibodies to confirm protein reduction and assess phenotypic consequences

• Ligand binding studies:

  • Express each receptor separately in heterologous systems

  • Use calcium imaging or cAMP assays to identify potential differential responses to ligands

• Protein interaction networks:

  • Perform immunoprecipitation using epitope-tagged versions of each receptor

  • Identify specific interaction partners through mass spectrometry analysis

• Signaling pathway analysis:

  • Assess downstream signaling activation (e.g., phosphorylation events) following receptor stimulation

  • Use phospho-specific antibodies in combination with OR10K1/OR10K2 detection

• Structural biology approaches:

  • Generate structural models based on sequence differences

  • Design experiments to test predicted functional differences in ligand binding pockets or G-protein coupling domains

What considerations should researchers take into account when designing multiplexed immunofluorescence panels including OR10K1/OR10K2 antibodies?

For successful multiplexed immunofluorescence including OR10K1/OR10K2 antibodies:

• Antibody compatibility assessment:

  • Ensure OR10K1/OR10K2 antibody host species (typically rabbit) is compatible with other antibodies in the panel

  • If using multiple rabbit antibodies, consider sequential immunostaining with direct conjugation or Fab fragment blocking between rounds

• Spectral considerations:

  • Select fluorophores with minimal spectral overlap for secondary antibodies

  • Include appropriate single-stain controls for spectral unmixing if using confocal or spectral imaging

• Antigen abundance balancing:

  • Adjust exposure times for each channel based on relative abundance of targets

  • OR10K1/OR10K2 may require longer exposure or signal amplification if expression is low compared to other targets

• Epitope retrieval optimization:

  • Test different antigen retrieval methods to ensure compatibility with all antibodies in the panel

  • Consider tyramide signal amplification for OR10K1/OR10K2 detection while maintaining compatibility with other retrieval methods

• Staining sequence determination:

  • Test different staining sequences to determine optimal order

  • Generally stain for lower abundance targets (potentially OR10K1/OR10K2) first

• Validation controls:

  • Include tissue microarrays or cell line controls with known expression patterns for all targets

  • Perform parallel single-marker staining to confirm multiplexed results

• Analysis considerations:

  • Develop quantitative image analysis protocols that account for potential differences in subcellular localization

  • Use colocalization analysis to investigate potential functional relationships with other markers

How does the differential expression of OR10K1 (downregulated) versus OR10K2 (upregulated) in thymic epithelial tumors inform potential research hypotheses?

The opposing regulation patterns of OR10K1 (downregulated) and OR10K2 (upregulated) in thymic epithelial tumors generate several research hypotheses:

• Functional antagonism hypothesis: These receptors may have opposing functions in tumor microenvironment regulation, with OR10K1 potentially serving as a tumor suppressor while OR10K2 functions as an oncogenic factor.

• Compensatory mechanism hypothesis: Upregulation of OR10K2 may represent a compensatory response to OR10K1 downregulation, suggesting overlapping but non-identical functions.

• Immune microenvironment modulation hypothesis: The differential expression may influence distinct aspects of immune cell recruitment, activation, or suppression within the tumor microenvironment.

• Prognostic biomarker development: The ratio of OR10K2 to OR10K1 expression might serve as a more robust prognostic indicator than either marker alone.

Research approaches to investigate these hypotheses include:

• Correlation analyses between receptor expression and clinical outcomes

• In vitro functional studies with receptor overexpression and knockdown

• Immune cell co-culture experiments to assess effects on immune function

• Integration with other components of the 11-gene prognostic model to understand pathway interactions

What experimental design would best address the discrepancy between calculated (35 kDa) and observed (72 kDa) molecular weights of OR10K1/OR10K2 proteins?

To investigate the molecular weight discrepancy between calculated (35 kDa) and observed (72 kDa) values for OR10K1/OR10K2 :

• Post-translational modification analysis:

  • Treatment with glycosidases (PNGase F, Endo H) to remove N-linked glycans

  • Phosphatase treatment to remove phosphate groups

  • Treatment with deubiquitinating enzymes to remove ubiquitin modifications

  • Western blot analysis after each treatment to assess shifts in molecular weight

• Expression system comparison:

  • Express recombinant OR10K1/OR10K2 with epitope tags in different systems (bacteria, insect cells, mammalian cells)

  • Compare molecular weights across expression systems with different post-translational modification capabilities

  • Use antibodies against both the epitope tag and OR10K1/OR10K2 to confirm identity

• Mass spectrometry analysis:

  • Immunoprecipitate OR10K1/OR10K2 from native sources

  • Perform LC-MS/MS analysis to identify the protein and characterize modifications

  • Quantify the stoichiometry of different modifications

• Membrane protein solubilization comparison:

  • Test different detergents and solubilization methods

  • Compare observed molecular weights under different denaturation conditions

  • Assess potential detergent-resistant oligomerization

• Domain deletion analysis:

  • Express truncated versions of the receptors lacking specific domains

  • Determine which regions contribute most significantly to the aberrant migration pattern

What new research applications might emerge from integrating OR10K1/OR10K2 antibody-based detection with other -omics approaches?

Integration of OR10K1/OR10K2 antibody-based detection with other -omics approaches offers several innovative research opportunities:

• Spatial transcriptomics integration:

  • Combine OR10K1/OR10K2 immunofluorescence with spatial transcriptomics to correlate protein expression with local transcriptional profiles

  • Map receptor distribution in relation to tissue microenvironments and signaling gradients

• Single-cell proteogenomics:

  • Integrate antibody-based flow cytometry for OR10K1/OR10K2 with single-cell RNA sequencing

  • Identify cell populations with discordant mRNA and protein expression patterns

• Interactome mapping:

  • Use proximity labeling approaches (BioID, APEX) with OR10K1/OR10K2 as baits

  • Identify tissue-specific protein interaction networks through mass spectrometry

  • Validate key interactions with co-immunoprecipitation using OR10K1/OR10K2 antibodies

• Clinical proteogenomics:

  • Correlate OR10K1/OR10K2 protein levels (by IHC or targeted proteomics) with genomic alterations in large patient cohorts

  • Develop integrated prognostic models incorporating both genetic and protein expression data

• Drug discovery applications:

  • Use OR10K1/OR10K2 antibodies in high-content screening to identify compounds that modulate receptor expression or localization

  • Combine with transcriptional profiling to understand mechanism of action

• Multi-modal imaging:

  • Develop correlative imaging workflows combining OR10K1/OR10K2 immunodetection with techniques like MALDI imaging mass spectrometry

  • Map receptor expression in relation to metabolite distributions in tissues