OR10J6P Antibody

What is OR10J6P and why are antibodies against it relevant for research?

OR10J6P (Olfactory Receptor, Family 10, Subfamily J, Member 6 Pseudogene) is a human pseudogene that was originally classified as part of the olfactory receptor family. Despite being a pseudogene, OR10J6P-derived proteins have been detected in certain human tissues, making antibodies against OR10J6P valuable for investigating its potential biological functions beyond olfaction. These antibodies enable researchers to detect endogenous levels of OR10J6P protein using techniques such as Western blotting, immunofluorescence, and ELISA .

The main applications of OR10J6P antibodies include:

• Protein expression profiling across different tissues

• Subcellular localization studies

• Investigation of potential roles in non-olfactory physiological processes

How does OR10J6P antibody specificity compare to other olfactory receptor antibodies?

OR10J6P antibodies are designed to detect specific epitopes, typically in the C-terminal region (amino acids 227-276) of the OR10J6P protein . This specificity is crucial when studying olfactory receptors due to the high sequence homology between family members.

When comparing OR10J6P antibodies with antibodies targeting related olfactory receptors like OR10G6, researchers should consider:

To ensure specificity, validation experiments using both positive controls (tissues/cells known to express OR10J6P) and negative controls (tissues/cells without OR10J6P expression) are recommended before proceeding with experimental applications .

What is the molecular profile of OR10J6P that researchers should be aware of?

Researchers working with OR10J6P antibodies should be familiar with key molecular characteristics of the target protein:

• Gene ID: 401973

• UniProt Accession: Q8NGY7

• Molecular Weight: Approximately 31 kDa

• Tissue Expression: Information about specific tissue expression patterns is limited, but the antibody has been optimized for detection in human samples

• Classification: Although designated as a pseudogene (OR10J6P), protein products have been detected in experimental settings, suggesting potential functional relevance

Understanding these characteristics helps researchers properly design experiments, accurately interpret results, and troubleshoot potential issues when working with OR10J6P antibodies.

What are the optimal experimental conditions for using OR10J6P antibodies in Western blotting?

For effective Western blot detection of OR10J6P protein, the following methodology is recommended based on validated protocols:

Sample Preparation:

• Use fresh tissue/cell lysates when possible

• Standard RIPA buffer with protease inhibitors is suitable for extraction

• Load 20-40 μg of total protein per lane

Western Blot Protocol:

• Use 10-12% SDS-PAGE gels for optimal resolution of the ~31 kDa target

• Transfer to PVDF membranes (recommended over nitrocellulose)

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature

• Primary antibody dilution: 1:500-1:1000 in blocking buffer

• Incubation: Overnight at 4°C with gentle rocking

• Secondary antibody: Anti-rabbit IgG conjugated with HRP at 1:5000-1:10000

• Detection: Standard ECL reagents are sufficient for visualization

Control experiments should include a loading control (e.g., GAPDH, β-actin) and, when possible, a positive control sample known to express OR10J6P protein. Western blot analysis has been successfully performed with these antibodies on lysates from COLO205 cells, which can serve as a positive control .

How should researchers optimize immunofluorescence protocols for OR10J6P detection?

For immunofluorescence applications using OR10J6P antibodies, researchers should follow these guidelines:

Fixation and Permeabilization:

• 4% paraformaldehyde fixation (10-15 minutes at room temperature)

• Permeabilization with 0.1-0.2% Triton X-100 (5-10 minutes)

• These conditions preserve epitope accessibility while maintaining cellular architecture

Staining Protocol:

• Blocking: 5% normal serum (goat or donkey) with 1% BSA in PBS (1 hour)

• Primary antibody: Dilute at 1:100-1:500 in blocking buffer

• Incubation: Overnight at 4°C in a humidified chamber

• Secondary antibody: Anti-rabbit IgG conjugated to a fluorophore (Alexa Fluor 488/594/647) at 1:200-1:500

• Counterstaining: DAPI (1:1000) for nuclear visualization

• Mounting: Anti-fade mounting medium to prevent photobleaching

For co-localization studies with other cellular markers, sequential staining with antibodies from different host species is recommended to prevent cross-reactivity. Confocal microscopy with appropriate filter settings should be used for optimal visualization and quantification of OR10J6P localization patterns.

What considerations are important when using OR10J6P antibodies for ELISA applications?

When developing ELISA protocols for OR10J6P detection, researchers should consider:

Antibody Dilution and Protocol Optimization:

• The recommended dilution for ELISA is 1:10000-1:40000, significantly more dilute than for other applications

• This dilution range should be validated with standard curves using recombinant OR10J6P protein if available

ELISA Format Selection:

• Indirect ELISA: Suitable for pure samples or recombinant proteins

• Sandwich ELISA: Required for complex samples like serum or tissue extracts

  • Requires pairs of OR10J6P antibodies recognizing different epitopes

  • Alternatively, pair with antibodies targeting fusion tags if using recombinant proteins

Optimization Parameters:

• Coating concentration: 1-5 μg/ml of capture antibody

• Blocking agent: 1-3% BSA or 5% non-fat milk

• Sample volume and incubation time: Typically 100 μl for 1-2 hours

• Detection system: HRP-labeled secondary antibody with TMB substrate

Standard curves should be generated using purified OR10J6P protein (if available) for accurate quantification, and all samples should be run in duplicate or triplicate to ensure reliability of results.

How can researchers address weak or absent signals when using OR10J6P antibodies?

When encountering weak or absent signals with OR10J6P antibodies, researchers should systematically evaluate:

Sample-related factors:

• Protein degradation: Ensure proper sample handling with fresh preparation and protease inhibitors

• Low target expression: OR10J6P may have tissue-specific or condition-dependent expression; confirm target presence using RT-PCR or other methods

• Improper protein extraction: Olfactory receptors are membrane proteins that may require specialized extraction buffers containing detergents

Protocol-related factors:

• Antibody dilution: If signal is weak, try more concentrated antibody (1:250-1:500 for WB/IF)

• Incubation conditions: Extended incubation times (up to 48h at 4°C) may enhance signal

• Detection sensitivity: Use enhanced chemiluminescent substrates or signal amplification systems

• Antigen retrieval: For fixed tissues, heat-induced or enzymatic antigen retrieval may be necessary

Experimental validation:

• Include positive controls known to express OR10J6P (e.g., COLO205 cells)

• Test antibody functionality with dot blots using the immunizing peptide

• Consider confirming results with an alternative OR10J6P antibody recognizing a different epitope

Maintaining detailed experimental records of optimization attempts will help identify the most effective conditions for specific research applications.

What are the common sources of non-specific binding with OR10J6P antibodies and how can they be minimized?

Non-specific binding is a common challenge when working with antibodies against olfactory receptors due to sequence homology within the family. To minimize these issues:

Causes and Solutions for Non-specific Binding:

For immunofluorescence applications specifically, autofluorescence can be reduced by including a quenching step (0.1% Sudan Black in 70% ethanol) before antibody application. Additionally, using highly cross-absorbed secondary antibodies helps minimize species cross-reactivity .

How should researchers validate OR10J6P antibody specificity for their particular experimental system?

Thorough validation of OR10J6P antibodies is essential before conducting experiments in new systems. A comprehensive validation approach includes:

Epitope-based validation:

• Peptide competition assays: Pre-incubation of antibody with the immunizing peptide (amino acids 227-276) should abolish specific signal

• Dot blot analysis with synthetic peptides corresponding to the target epitope

Expression-based validation:

• Positive and negative controls: Test the antibody in cell lines/tissues with known OR10J6P expression status

• siRNA/shRNA knockdown: Reduction of target protein should correspond with decreased antibody signal

• Overexpression: Transient transfection with OR10J6P expression constructs should show increased signal intensity

Specificity assessment:

• Western blot should show a predominant band at the expected molecular weight (~31 kDa)

• Immunofluorescence pattern should be consistent with the expected subcellular localization

• Mass spectrometry confirmation of immunoprecipitated proteins can provide definitive validation

Documentation of validation results is crucial for publication and reproducibility purposes, particularly given the challenges associated with antibodies targeting olfactory receptor family members.

What are the key considerations for using OR10J6P antibodies in high-throughput screening and developability assessments?

OR10J6P antibodies can be incorporated into high-throughput screening workflows, similar to those described for therapeutic antibody development. Key considerations include:

Assay Miniaturization and Automation:

• Reduce reaction volumes to 20-50 μl for 384-well format compatibility

• Optimize antibody concentrations to conserve reagents while maintaining signal-to-noise ratio

• Implement automated liquid handling systems for consistent reagent dispensing

Multiplexed Detection Systems:

• Pair OR10J6P antibodies with other markers for co-detection using:

  • Multiplex immunofluorescence with spectrally distinct fluorophores

  • Sequential chromogenic immunohistochemistry

  • Bead-based multiplex assay systems

Quality Control Metrics:

• Z'-factor > 0.5 indicates robust assay performance

• Signal-to-background ratio > 5:1 for reliable detection

• Coefficient of variation < 20% across replicates

For developability assessments, researchers should evaluate antibody stability and performance under various conditions as outlined in the high-throughput developability workflow described for therapeutic antibodies . This includes thermal stability, colloidal stability, and potential for self-interaction which can be assessed using techniques like differential scanning fluorimetry and dynamic light scattering.

How can researchers utilize OR10J6P antibodies in combination with modern -omics approaches?

Integration of OR10J6P antibody-based detection with -omics technologies offers powerful insights into the biological context of this protein:

Immunoprecipitation-Mass Spectrometry (IP-MS):

• Use OR10J6P antibodies for immunoprecipitation followed by mass spectrometry to:

  • Identify interaction partners in different cellular contexts

  • Characterize post-translational modifications on OR10J6P

  • Confirm antibody specificity through peptide identification

ChIP-Seq Applications:

• If OR10J6P has nuclear functions (uncommon but possible for some olfactory receptors):

  • OR10J6P antibodies can be used for chromatin immunoprecipitation

  • Followed by sequencing to identify potential DNA binding sites

Spatial Transcriptomics Integration:

• Correlate OR10J6P protein expression (via immunohistochemistry) with:

  • Spatial transcriptomics data in the same tissue sections

  • Single-cell RNA-seq profiles to identify cell populations expressing OR10J6P

Bioinformatic Analysis Workflow:

• Raw data acquisition (protein quantification, localization patterns)

• Quality control and normalization

• Integration with -omics datasets (transcriptomics, proteomics)

• Pathway and network analysis to contextualize OR10J6P function

• Visualization using tools like Cytoscape for protein interaction networks

These integrated approaches provide context for understanding OR10J6P's potential roles beyond conventional olfactory functions .

What methodological approaches should be employed when investigating OR10J6P in non-traditional tissues beyond the olfactory system?

Recent research suggests olfactory receptors may have functions beyond their canonical roles in olfaction. When investigating OR10J6P in non-traditional tissues:

Tissue-Specific Protocol Modifications:

Experimental Design for Functional Characterization:

• Parallel protein and mRNA detection to confirm expression

• Co-localization with tissue-specific markers to identify expressing cell types

• Functional assays relevant to the tissue context (e.g., calcium signaling, cAMP production)

• Genetic manipulation (siRNA, CRISPR) to assess functional consequences of OR10J6P modulation

Controls and Validation:

• Include established olfactory tissue as positive control

• Use secondary-only and isotype controls to rule out non-specific binding

• Confirm specificity through peptide competition assays in each new tissue type

• Consider orthogonal detection methods (RNAscope, in situ hybridization) to confirm expression patterns

These approaches help establish whether OR10J6P has legitimate functions in non-olfactory tissues while minimizing the risk of artifacts or misinterpretation of results .

What future research directions might benefit from OR10J6P antibodies?

OR10J6P antibodies are valuable tools for exploring several emerging research areas:

• Ectopic Expression in Disease States: Investigating potential dysregulation of OR10J6P in pathological conditions such as cancer, neurodegeneration, or inflammatory diseases

• Drug Discovery Applications: Screening for compounds that modulate OR10J6P expression or function in relevant cellular contexts

• Developmental Biology: Tracking OR10J6P expression during embryonic and postnatal development to identify potential roles in tissue differentiation

• Comparative Biology: Examining OR10J6P homologs across species to understand evolutionary conservation and divergence of function

• Single-Cell Resolution Studies: Combining OR10J6P antibodies with single-cell techniques to identify rare cell populations expressing this protein