OR2M2 Antibody

What is OR2M2 and why is it studied in research?

OR2M2 (Olfactory receptor 2M2, also known as OST423) is a member of the G-protein coupled receptor 1 family functioning as an odorant receptor. As a transmembrane protein with seven domains, it plays a critical role in the olfactory transduction pathway by initiating neuronal responses that trigger smell perception. Research on OR2M2 is valuable for understanding sensory biology, neuroscience, and potential applications in food science and fragrance development. The protein contains 201 amino acid residues with a molecular weight of approximately 39 kDa and is primarily located in the cell membrane as a multi-pass membrane protein .

What are the common applications for OR2M2 antibodies in research?

OR2M2 antibodies are utilized in multiple research techniques, with the most common applications being:

These applications enable researchers to detect endogenous levels of OR2M2 protein in various sample types, making them valuable tools for olfactory system studies .

What are the key differences between polyclonal and monoclonal antibodies against OR2M2?

How should I optimize Western blot conditions for OR2M2 detection?

For optimal OR2M2 detection via Western blot:

• Sample preparation: Extract proteins from tissues or cells using a buffer containing protease inhibitors to prevent degradation. OR2M2 is membrane-associated, so consider using detergent-based lysis buffers (e.g., RIPA with 0.1% SDS).

• Gel selection: Use 10-12% SDS-PAGE gels as OR2M2 has a predicted molecular weight of 39 kDa, though observed weight may vary (24-28 kDa, 41-47 kDa) due to post-translational modifications.

• Transfer conditions: Transfer to PVDF membranes (preferred over nitrocellulose) at 100V for 1 hour or 30V overnight at 4°C.

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

• Antibody dilution: Dilute primary OR2M2 antibody 1:500-1:1000 in blocking buffer and incubate overnight at 4°C.

• Detection system: Use HRP-conjugated secondary antibodies and enhanced chemiluminescence for detection.

• Controls: Include positive controls (e.g., L02 cells, A549 cells) which are known to express OR2M2 .

What are the recommended protocols for immunohistochemical detection of OR2M2?

For successful immunohistochemical detection of OR2M2:

• Tissue preparation: Fix tissues in 10% neutral buffered formalin and embed in paraffin. Cut sections at 4-6 μm thickness.

• Antigen retrieval: This step is critical - use TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0 in a pressure cooker or water bath at 95-98°C for 20 minutes.

• Blocking: Block endogenous peroxidase with 3% H₂O₂ for 10 minutes, then block non-specific binding with 5-10% normal serum.

• Primary antibody: Dilute OR2M2 antibody 1:50-1:500 (optimize for your specific antibody) and incubate overnight at 4°C.

• Detection system: Use appropriate HRP/DAB detection systems according to the manufacturer's protocol.

• Counterstaining: Counterstain with hematoxylin for nuclear visualization.

• Positive control: Human liver tissue has been validated for OR2M2 detection.

• Negative control: Include a section with isotype control antibody .

How can I validate the specificity of my OR2M2 antibody?

Validating OR2M2 antibody specificity requires multiple complementary approaches:

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide (if available) before application to your sample. Signal elimination confirms specificity.

• Knockout/knockdown controls: Compare OR2M2 detection in wild-type versus OR2M2 knockout or siRNA-treated samples. Reduced signal in knockdown samples confirms specificity.

• Multiple antibodies: Use multiple antibodies raised against different epitopes of OR2M2 and compare detection patterns.

• Recombinant protein: Test antibody against purified recombinant OR2M2 protein to confirm binding.

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein.

• Cross-reactivity testing: Test the antibody against closely related proteins, particularly other olfactory receptors.

• Cellular localization: Confirm that immunostaining matches the expected membrane localization pattern for a G-protein coupled receptor .

Why might I observe multiple bands when detecting OR2M2 by Western blot?

Multiple bands in OR2M2 Western blots may occur for several reasons:

• Post-translational modifications: OR2M2 undergoes N-glycosylation, which can result in bands of higher molecular weight (41-47 kDa) than the calculated mass (39 kDa).

• Protein degradation: Incomplete protease inhibition may lead to degradation products appearing as lower molecular weight bands.

• Isoforms: While not extensively documented for OR2M2, potential splice variants could result in proteins of different sizes.

• Oligomerization: GPCR proteins like OR2M2 can form dimers or higher-order oligomers that may not completely dissociate under standard SDS-PAGE conditions.

• Cross-reactivity: The antibody may recognize structurally similar olfactory receptors, particularly if using polyclonal antibodies.

To address these issues, include positive controls with known OR2M2 expression, optimize sample preparation to minimize degradation, and consider deglycosylation experiments if glycosylation is suspected as the cause of multiple bands .

How can I improve signal-to-noise ratio when using OR2M2 antibodies in immunofluorescence?

To improve signal-to-noise ratio in OR2M2 immunofluorescence:

• Fixation optimization: Test different fixation methods; 4% paraformaldehyde for 10-15 minutes is often optimal for membrane proteins like OR2M2.

• Permeabilization: For intracellular epitopes, use 0.1-0.3% Triton X-100 or 0.1% saponin. Over-permeabilization can increase background.

• Blocking: Extend blocking time to 1-2 hours using 5-10% normal serum from the species of your secondary antibody. Consider adding 0.1-0.3% Triton X-100 to the blocking solution.

• Antibody dilution: Test a range of antibody dilutions (1:100-1:800) to find the optimal concentration that maximizes specific signal while minimizing background.

• Incubation conditions: Incubate primary antibody overnight at 4°C with gentle agitation.

• Washing steps: Increase the number and duration of washing steps (at least 3×10 minutes with PBS-T).

• Antigen retrieval: For fixed tissues or cells, optimize antigen retrieval methods to enhance epitope accessibility.

• Controls: Include negative controls (secondary antibody only) and positive controls (cells known to express OR2M2, such as HEK-293 cells) .

How should I interpret contradictory results between different OR2M2 antibody applications?

When facing contradictory results across different applications of OR2M2 antibodies:

• Epitope accessibility: Different applications expose different epitopes. In Western blot, proteins are denatured, exposing all linear epitopes, while in immunofluorescence or IHC, proteins retain much of their native conformation, potentially masking some epitopes.

• Antibody characteristics: Some antibodies work well in one application but poorly in others due to their specific binding properties. Check if your antibody has been validated for your specific application.

• Protein abundance threshold: Different techniques have different detection thresholds. Western blot can be more sensitive than IHC for low-abundance proteins.

• Sample preparation differences: Fixation, embedding, and antigen retrieval methods in IHC/IF can affect epitope preservation differently than sample preparation for Western blot.

• Cross-reactivity in complex samples: An antibody might cross-react with other proteins in complex samples but show specificity in purified systems.

To resolve contradictions, validate results using complementary techniques and multiple antibodies recognizing different epitopes of OR2M2 .

How can OR2M2 antibodies be utilized in studying olfactory signal transduction pathways?

OR2M2 antibodies can be powerful tools for investigating olfactory signal transduction:

• Co-immunoprecipitation (Co-IP): Use OR2M2 antibodies to pull down OR2M2 and associated proteins to identify interaction partners in the signaling cascade, particularly G-proteins and downstream effectors.

• Chromatin immunoprecipitation (ChIP): Apply OR2M2 antibodies in ChIP assays to investigate potential transcription factors that regulate OR2M2 expression.

• Proximity ligation assay (PLA): Combine OR2M2 antibodies with antibodies against potential interacting proteins to visualize and quantify protein-protein interactions in situ.

• Super-resolution microscopy: Use immunofluorescence with OR2M2 antibodies in techniques like STORM or PALM to examine the nanoscale distribution and clustering of OR2M2 receptors in membranes.

• Tissue-specific expression profiling: Employ OR2M2 antibodies in multi-label immunofluorescence to map the expression patterns of OR2M2 across different olfactory neuron subtypes.

• Functional studies: Combine OR2M2 antibody labeling with calcium imaging to correlate receptor expression with functional responses to specific odorants .

What approaches can be used to study post-translational modifications of OR2M2 using specific antibodies?

To investigate post-translational modifications (PTMs) of OR2M2:

• Modification-specific antibodies: Use antibodies specifically recognizing phosphorylated, glycosylated, or ubiquitinated forms of OR2M2, if available.

• Two-dimensional electrophoresis: Combine with Western blotting using OR2M2 antibodies to separate proteins by both isoelectric point and molecular weight, revealing different modified forms.

• Immunoprecipitation-mass spectrometry: Immunoprecipitate OR2M2 using specific antibodies and analyze by mass spectrometry to identify and characterize PTMs.

• Glycosidase treatment: Treat samples with enzymes like PNGase F before Western blotting to remove N-linked glycans and observe mobility shifts.

• Phosphatase treatment: Compare phosphatase-treated versus untreated samples to identify phosphorylation-dependent mobility shifts.

• Site-directed mutagenesis: Combine with antibody detection to confirm specific modification sites (e.g., S291 phosphorylation site mentioned in search results).

• Dynamic studies: Use pulse-chase labeling combined with immunoprecipitation to study the kinetics of OR2M2 modifications .

How can OR2M2 antibodies contribute to studying genetic variation in olfactory receptors?

OR2M2 antibodies can help investigate genetic variation in olfactory receptors through:

• Allele-specific expression: Use antibodies in conjunction with genotyping to determine if genetic variants affect protein expression levels in heterozygous samples.

• Functional consequences of variants: Compare protein localization, stability, or trafficking between wild-type and variant forms using immunofluorescence or Western blotting.

• Population studies: Apply antibodies in tissue microarrays from diverse populations to correlate OR2M2 protein expression with known genetic variants.

• Cross-species comparison: Use antibodies with cross-reactivity to mouse, rat, or other species (as mentioned in search results) to study evolutionary conservation of protein expression patterns.

• Haplotype studies: Combine antibody detection with haplotype analysis to investigate if certain combinations of variants affect protein expression or function.

• Linkage disequilibrium analysis: Investigate if variants in OR2M2 that affect protein detection are in linkage disequilibrium with variants in other genes, potentially revealing functional gene clusters .

What is the extent of cross-reactivity between OR2M2 antibodies and other olfactory receptors?

Cross-reactivity of OR2M2 antibodies with other olfactory receptors is an important consideration:

• Sequence homology: The human genome contains approximately 400 functional olfactory receptor genes with significant sequence homology. OR2M2 belongs to family 2, subfamily M, which contains multiple members with similar sequences.

• Epitope selection: Antibodies raised against conserved regions of OR2M2 are more likely to cross-react with related olfactory receptors. Antibodies targeting unique regions (like the C-terminal region mentioned in some products) may offer greater specificity.

• Validation studies: Few comprehensive cross-reactivity studies exist specifically for OR2M2 antibodies. Most commercial antibodies report specificity for OR2M2 but may not have been tested against all related olfactory receptors.

• Experimental validation: Researchers should consider testing specificity using overexpression systems with related olfactory receptors or using siRNA knockdown of OR2M2 to confirm antibody specificity.

• Computational prediction: Epitope mapping and sequence alignment can help predict potential cross-reactivity with other olfactory receptors .

How should researchers account for species differences when using OR2M2 antibodies?

When using OR2M2 antibodies across different species:

• Sequence conservation: While human OR2M2 shares homology with mouse and rat orthologs, there are significant sequence differences. Human antibodies may not recognize the same epitopes in other species.

• Validated species reactivity: Refer to the table below showing reported species reactivity for commercial OR2M2 antibodies:

• Epitope conservation analysis: Before using a human OR2M2 antibody in another species, align the immunogen sequence with the target species' sequence to assess epitope conservation.

• Validation requirements: Additional validation steps are necessary when using antibodies across species, including positive controls from the target species and comparison with species-specific antibodies if available.

• Alternative approaches: For cross-species studies, consider using epitope tags (FLAG, HA, etc.) with OR2M2 expression constructs rather than relying solely on direct antibody detection .

What are the challenges in generating highly specific antibodies against OR2M2?

Generating highly specific OR2M2 antibodies faces several challenges:

• Protein family similarity: OR2M2 belongs to the large olfactory receptor family with high sequence similarity among members, making unique epitope identification difficult.

• Membrane protein nature: As a seven-transmembrane G-protein coupled receptor, OR2M2 is difficult to purify in its native conformation, limiting the use of whole-protein immunization.

• Expression levels: Natural expression levels of OR2M2 are relatively low, making it challenging to purify sufficient quantities for immunization.

• Post-translational modifications: N-glycosylation and other modifications can affect epitope accessibility and antibody recognition.

• Peptide design limitations: Most commercial antibodies use synthetic peptides derived from predicted antigenic regions, but these may not represent the protein's native conformation.

• Validation complexity: Comprehensive validation requires testing against all related olfactory receptors, which is practically challenging given the large number (~400) of olfactory receptor genes.

• Species conservation: Generating antibodies that work across species requires targeting highly conserved epitopes, which may coincidentally be conserved in other olfactory receptors, creating a specificity-cross-reactivity tradeoff .