OR5M1/OR5M10 Antibody

What are the primary validated applications for OR5M1/OR5M10 antibodies?

OR5M1/OR5M10 antibodies have been validated for several key applications in molecular and cellular biology research. The primary validated applications include Western Blotting (WB), Enzyme-Linked Immunosorbent Assay (ELISA), and Immunofluorescence/Immunocytochemistry (IF/ICC) . When planning experiments, researchers should consider that most commercial antibodies show optimal performance in WB at dilutions ranging from 1:500-1:2000, while ELISA applications typically require higher dilutions (1:5000-1:20000) . For immunofluorescence applications, a concentration range of 1:100-1:500 is generally recommended . These application-specific dilutions are critical starting points that may require optimization based on your specific experimental conditions and sample type.

How should OR5M1/OR5M10 antibodies be stored to maintain optimal activity?

Proper storage is crucial for maintaining antibody activity. OR5M1/OR5M10 antibodies should be stored at -20°C for long-term preservation (up to one year) . For frequent use and short-term storage (up to one month), 4°C is appropriate . It is essential to aliquot the antibody upon receipt to minimize freeze-thaw cycles, as repeated freezing and thawing can significantly reduce antibody effectiveness . Most commercial preparations are supplied in a stabilizing buffer containing 50% glycerol and 0.02% sodium azide, which helps maintain antibody integrity during storage . When handling the antibody, researchers should note that sodium azide is toxic and requires appropriate safety precautions .

What is the molecular basis for OR5M1/OR5M10 detection in experimental systems?

OR5M1 and OR5M10 are olfactory receptors belonging to the G-protein-coupled receptor (GPCR) family . These receptors feature a characteristic 7-transmembrane domain structure shared with many neurotransmitter and hormone receptors . OR5M1/OR5M10 antibodies typically recognize specific epitopes within these proteins, with immunogens often derived from the C-terminal region or internal sequences (amino acids 30-110, 155-204, 220-279, or 240-289, depending on the specific antibody) . The calculated molecular weight of these targets is approximately 35-36 kDa , which serves as an important reference point when validating antibody specificity in Western blot applications. Understanding these molecular characteristics is essential for proper experimental design and interpretation of results.

How can researchers validate the specificity of OR5M1/OR5M10 antibodies in their experimental system?

Validating antibody specificity is a critical step before proceeding with experiments. For OR5M1/OR5M10 antibodies, a multi-faceted approach is recommended:

• Positive control selection: Use cell lines known to express OR5M1/OR5M10, such as Jurkat cells for Western blot applications or MCF7 cells for immunofluorescence studies .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples. This should abolish specific staining, as demonstrated in validation studies with MCF7 cells .

• Molecular weight verification: Confirm that the detected band in Western blot corresponds to the expected molecular weight of approximately 35-36 kDa .

• Negative controls: Include samples where primary antibody is omitted or replaced with non-specific IgG from the same species.

• Cross-reactivity assessment: If working with non-human samples, verify cross-reactivity claims, noting that most OR5M1/OR5M10 antibodies are primarily validated for human samples .

This comprehensive validation ensures that experimental results are attributable to specific antibody-antigen interactions rather than non-specific binding.

What are the optimal sample preparation protocols for detecting OR5M1/OR5M10 in different experimental contexts?

Optimal sample preparation varies by application:

For Western Blot:

• Lyse cells in a buffer containing protease inhibitors to prevent degradation of target proteins

• Typical protein concentrations range from 20-50 μg per lane

• Use fresh samples when possible, as OR5M1/OR5M10 may degrade during storage

• Jurkat cells have been successfully used as positive controls

For Immunofluorescence:

• Fix cells with 4% paraformaldehyde for 15-20 minutes at room temperature

• For membrane proteins like OR5M1/OR5M10, mild permeabilization with 0.1-0.2% Triton X-100 is recommended

• Block with 1-5% BSA or normal serum from the secondary antibody host species

• MCF7 cells have been validated as appropriate for OR5M1 immunofluorescence studies

For ELISA:

• Coating concentration should be optimized for each specific antibody

• The high dilution range (1:5000-1:20000) for polyclonal OR5M1/OR5M10 antibodies in ELISA applications indicates good sensitivity

These protocols provide starting points that may require further optimization based on specific experimental requirements.

How do the different epitope regions targeted by various OR5M1/OR5M10 antibodies affect experimental outcomes?

Commercial OR5M1/OR5M10 antibodies target different epitope regions, which can significantly impact experimental results:

Researchers should select antibodies whose target epitopes are accessible in their experimental system. For instance, if the C-terminal region is involved in protein-protein interactions in your experimental context, antibodies targeting other regions might provide more consistent results. Additionally, epitope masking due to protein folding or post-translational modifications can affect antibody binding, so using antibodies targeting different regions may be advisable when contradictory results are observed.

What strategies can researchers employ when experiencing weak or variable signal intensity with OR5M1/OR5M10 antibodies?

When encountering weak or inconsistent signals with OR5M1/OR5M10 antibodies, consider these methodological solutions:

• Signal Amplification: For immunofluorescence applications showing weak signals, implement tyramide signal amplification (TSA) or use brightfield detection with DAB substrate and nickel enhancement.

• Epitope Retrieval Optimization: GPCR epitopes may be masked by fixation or sample preparation. Test different antigen retrieval methods, such as:

  • Heat-mediated retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Enzymatic retrieval using proteinase K (1-5 μg/ml for 5-15 minutes)

  • For Western blots, adjust SDS concentration or heating time during sample preparation

• Concentration Gradient Testing: Perform a systematic titration of antibody concentrations beyond the recommended range (e.g., 1:100 to 1:5000) to determine the optimal signal-to-noise ratio for your specific sample type.

• Alternative Detection Systems: If using chemiluminescence for Western blots, switch to more sensitive substrates or consider fluorescent secondary antibodies for quantitative analysis.

• Sample Enrichment: For low-abundance targets like OR5M1/OR5M10, implement subcellular fractionation to enrich membrane proteins before Western blotting or immunoprecipitation.

These approaches should be systematically tested and documented to identify the optimal conditions for your specific experimental system.

How can researchers distinguish between OR5M1 and OR5M10 detection when using antibodies that recognize both proteins?

Distinguishing between OR5M1 and OR5M10 presents a challenge as many commercial antibodies recognize both proteins due to their high sequence homology. Consider these methodological approaches:

• RNA interference: Design specific siRNAs targeting unique regions of OR5M1 or OR5M10 mRNAs. Compare antibody signal patterns between control and knockdown samples to determine the contribution of each protein to the observed signal.

• Bioinformatic analysis: Analyze the immunogen sequences used to generate the antibody and compare them to OR5M1 and OR5M10 sequences to identify potential differential binding affinities.

• Recombinant protein controls: Express OR5M1 and OR5M10 recombinant proteins separately and use them as controls in Western blots to establish migration patterns that might distinguish the two proteins.

• High-resolution techniques: Employ 2D gel electrophoresis followed by Western blotting to potentially separate OR5M1 and OR5M10 based on both size and isoelectric point differences.

• Complementary detection methods: Combine antibody detection with mass spectrometry analysis to identify peptide fragments unique to either OR5M1 or OR5M10.

When absolute discrimination is critical, researchers might need to employ multiple approaches simultaneously and validate findings through independent methodologies.

What are the considerations for integrating OR5M1/OR5M10 antibody detection with other advanced research techniques?

Integrating OR5M1/OR5M10 antibody detection with advanced research techniques requires careful methodological planning:

For Co-immunoprecipitation and Protein Interaction Studies:

• Use mild lysis conditions (e.g., 1% NP-40 or 0.5% Triton X-100) to preserve protein-protein interactions

• Consider crosslinking approaches for transient interactions

• Validate antibody specificity in IP applications separately, as not all WB-validated antibodies perform well in IP

For Super-resolution Microscopy:

• Select antibodies with minimal background and high specificity

• Consider directly conjugated primary antibodies to avoid spatial displacement caused by secondary antibodies

• Validate signal specificity with peptide competition assays or knockout controls

For Flow Cytometry:

• Test fixation and permeabilization protocols specifically for membrane proteins

• Titrate antibody concentrations to minimize background while maintaining specific signal

• Consider fluorophore brightness and spectral overlap when designing multi-parameter panels

For In vivo Imaging:

• Evaluate antibody specificity in the target tissue using immunohistochemistry before proceeding to in vivo applications

• Consider pharmacokinetics and biodistribution of antibody-based probes

• Test for immunogenicity if planning longitudinal studies

These methodological considerations ensure that OR5M1/OR5M10 antibodies can be effectively integrated into advanced research workflows while maintaining experimental rigor and reproducibility.

How should researchers interpret OR5M1/OR5M10 expression patterns in non-olfactory tissues?

Interpreting OR5M1/OR5M10 expression in non-olfactory tissues requires careful consideration of biological and technical factors:

• Biological significance: While traditionally associated with olfactory neurons, ectopic expression of olfactory receptors has been documented in various tissues. This may represent:

  • Functional repurposing in non-olfactory contexts

  • Involvement in non-canonical signaling pathways

  • Developmental remnants with unclear functional significance

• Technical validation approach:

  • Confirm specificity with multiple detection methods (e.g., RNA-seq, RT-PCR, in situ hybridization)

  • Use appropriate positive controls (olfactory epithelium) and negative controls

  • Quantify expression levels relative to olfactory tissue to assess physiological relevance

• Functional assessment strategies:

  • Investigate whether canonical OR signaling components (Gαolf, ACIII) are co-expressed

  • Conduct loss-of-function experiments to determine phenotypic effects

  • Identify potential ligands in the non-olfactory context through screening approaches

• Interpretation caveats:

  • Low expression levels may still be functionally significant

  • Antibody cross-reactivity with related GPCRs must be ruled out

  • Cell-type specific expression may be diluted in whole-tissue analyses

This systematic approach helps researchers distinguish between biologically relevant expression and technical artifacts when investigating OR5M1/OR5M10 in non-canonical contexts.

What factors explain contradictory results between different OR5M1/OR5M10 antibodies in the same experimental system?

When different OR5M1/OR5M10 antibodies yield contradictory results, consider these methodological explanations:

• Epitope accessibility variation:

  • Different antibodies target distinct regions of OR5M1/OR5M10 proteins

  • Protein conformational changes may mask specific epitopes

  • Post-translational modifications may block antibody binding sites

  • Protein-protein interactions may conceal certain regions

• Methodological differences:

  • Fixation protocols can differentially affect epitope preservation

  • Sample preparation methods may impact protein conformation

  • Buffer compositions can influence antibody-antigen interactions

  • Incubation times and temperatures affect binding kinetics

• Antibody characteristics:

  • Polyclonal preparations contain multiple antibody species with varying affinities

  • Lot-to-lot variations may exist, especially in polyclonal antibodies

  • Some antibodies may recognize denatured epitopes more effectively than native forms

• Resolution strategy:

  • Implement a systematic comparison using standardized protocols

  • Include appropriate positive and negative controls

  • Use complementary detection methods (e.g., RNA analysis)

  • Consider epitope mapping to identify the precise binding regions

Understanding these factors allows researchers to reconcile contradictory findings and develop more robust experimental approaches for OR5M1/OR5M10 detection.

How does the expression and function of OR5M1/OR5M10 relate to broader research contexts in GPCR biology?

OR5M1/OR5M10 research contributes significantly to the broader context of GPCR biology:

• Evolutionary perspectives:

  • Olfactory receptors represent the largest GPCR subfamily in mammals

  • Study of OR5M1/OR5M10 provides insights into GPCR evolution and diversification

  • Comparative analysis across species informs understanding of receptor specialization

• Structural implications:

  • ORs share the canonical 7-transmembrane domain structure with other GPCRs

  • Insights from OR5M1/OR5M10 binding mechanisms may inform structure-function relationships in other GPCRs

  • Differences in ligand binding domains contribute to our understanding of GPCR selectivity

• Signaling pathway integration:

  • OR5M1/OR5M10 utilize G-protein coupled signaling similar to other GPCRs

  • Cross-talk between OR signaling and other cellular pathways informs signal integration models

  • Understanding OR desensitization mechanisms contributes to broader GPCR regulation concepts

• Pharmacological relevance:

  • OR5M1/OR5M10 research methodology parallels approaches used in drug discovery for other GPCR targets

  • Ligand screening approaches for ORs inform general GPCR targeting strategies

  • Ectopic expression patterns suggest potential roles in non-olfactory processes with therapeutic implications

This contextual understanding positions OR5M1/OR5M10 research within the larger framework of GPCR biology, providing both specific insights into olfactory function and broader implications for receptor biology.

What methodological approaches can determine the functional significance of OR5M1/OR5M10 in experimental systems?

To establish functional significance of OR5M1/OR5M10, researchers should implement these methodological strategies:

• Genetic perturbation techniques:

  • CRISPR/Cas9-mediated knockout or knockin models

  • Inducible expression systems for temporal control

  • siRNA or shRNA for transient suppression

  • Assess phenotypic consequences across multiple cellular processes

• Ligand identification:

  • High-throughput screening of odorant libraries

  • Calcium imaging to detect receptor activation

  • cAMP accumulation assays for downstream signaling

  • Structure-activity relationship studies to map binding requirements

• Signaling pathway characterization:

  • Phosphorylation studies of downstream effectors

  • Protein-protein interaction mapping via proximity labeling

  • Temporal analysis of signaling dynamics

  • Pathway inhibitor studies to establish necessity and sufficiency

• Translational approaches:

  • Correlation of receptor expression with physiological or pathological states

  • Population-based genetic association studies

  • Ex vivo functional assays using patient-derived samples

  • Animal models with orthologous receptor modifications

These complementary approaches provide robust evidence for functional roles beyond mere expression data, establishing causal relationships between OR5M1/OR5M10 activity and biological processes.

How can researchers integrate OR5M1/OR5M10 antibody-based detection with single-cell analysis techniques?

Integrating OR5M1/OR5M10 antibody detection with single-cell technologies requires careful methodological considerations:

• Single-cell Western blotting:

  • Microfluidic platforms can separate individual cells before protein analysis

  • Requires high antibody specificity due to limited sample amount

  • Consider signal amplification strategies to detect low-abundance targets

  • Multiplexing with other protein markers enables cellular heterogeneity assessment

• Mass cytometry (CyTOF):

  • Conjugate OR5M1/OR5M10 antibodies with isotopically pure metals

  • Validate specificity and sensitivity of metal-conjugated antibodies

  • Optimize staining protocols for membrane proteins

  • Design panels including lineage markers and functional readouts

• Imaging mass cytometry:

  • Enables spatial visualization of OR5M1/OR5M10 in tissue context

  • Requires careful antibody validation for tissue applications

  • Consider antigen retrieval optimization for fixed tissues

  • Multiplexing capacity allows contextual protein interaction analysis

• Single-cell multi-omics integration:

  • Combine antibody-based protein detection with transcriptomics

  • CITE-seq approaches can correlate surface protein expression with gene expression

  • Consider computational methods for integrating protein and RNA data

  • Trajectory analysis can reveal dynamic regulation of OR5M1/OR5M10

These approaches enable researchers to move beyond population averages, revealing cell-specific expression patterns and functional heterogeneity of OR5M1/OR5M10 in complex biological systems.

What considerations are important when developing customized OR5M1/OR5M10 antibodies for specialized research applications?

When developing customized OR5M1/OR5M10 antibodies for specialized applications, researchers should consider these methodological aspects:

• Epitope selection strategy:

  • Analyze sequence conservation across species if cross-reactivity is desired

  • Assess hydrophilicity, surface probability, and antigenicity profiles

  • Target regions unique to OR5M1 or OR5M10 if discrimination is required

  • Avoid transmembrane domains which may be inaccessible in native proteins

  • Optimal epitope length is typically 10-20 amino acids

• Immunization protocol optimization:

  • Consider multiple host species to overcome tolerance to conserved mammalian epitopes

  • Implement prime-boost strategies with different adjuvants

  • Monitor antibody titers throughout immunization

  • Screen serum against both immunogen and native protein

• Affinity purification considerations:

  • Compare antigen-specific purification with protein A/G approaches

  • Assess elution conditions impact on antibody activity

  • Consider negative selection against related olfactory receptors

  • Validate specificity against recombinant OR5M1 and OR5M10

• Functional modification options:

  • Enzymatic digestion to generate Fab fragments for improved tissue penetration

  • Site-specific conjugation methods to preserve antigen-binding regions

  • Consider recombinant antibody production for reproducibility

  • Engineer affinity tags for purification or detection without compromising binding