OR5M1 Antibody

What is OR5M1 and why is it studied in research?

OR5M1 (Olfactory Receptor Family 5 Subfamily M Member 1) is a protein that functions as an odorant receptor. It belongs to the G-protein coupled receptor 1 family and is primarily localized in the cell membrane as a multi-pass membrane protein . Olfactory receptors like OR5M1 interact with odorant molecules in the nose to initiate neuronal responses that trigger smell perception . With a molecular weight of approximately 35.6 kDa, OR5M1 is encoded by the gene ID 390168 and has the UniProt accession number Q8NGP8 . Research on OR5M1 contributes to our understanding of olfactory signal transduction pathways and may have implications for sensory neuroscience.

What types of OR5M1 antibodies are currently available for research?

Currently available OR5M1 antibodies are predominantly rabbit polyclonal antibodies that target different epitopes of the protein. The most common types include:

Most of these antibodies are generated by immunizing rabbits with KLH-conjugated synthetic peptides derived from specific regions of human OR5M1 . They are typically purified using protein A or peptide affinity chromatography techniques .

What is the difference between OR5M1 and OR5M1/OR5M10 dual-specificity antibodies?

Some commercially available antibodies are designed to recognize both OR5M1 and the closely related OR5M10 protein. This distinction is important for experimental design:

• OR5M1-specific antibodies: Target unique epitopes in OR5M1, typically from the C-terminal region (amino acids 244-272) . These provide higher specificity for OR5M1 alone.

• OR5M1/OR5M10 dual-specificity antibodies: Recognize epitopes common to both proteins, usually targeting the region between amino acids 220-269 . These antibodies can detect both proteins simultaneously, which may be advantageous for studying this receptor subfamily but may complicate interpretation when specificity to a single protein is required .

Researchers should carefully select the appropriate antibody based on their experimental needs, considering whether they require detection of OR5M1 specifically or both OR5M1 and OR5M10 proteins .

Experimental Applications and Methodological Approaches

Optimizing Western blot protocols for OR5M1 detection requires attention to several methodological aspects:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • Consider membrane protein extraction protocols for optimal OR5M1 solubilization, as it is a multi-pass membrane protein

  • Load 30-35 μg of total protein per lane based on successful detection in validation studies

• Antibody dilution optimization:

  • Start with a 1:1000 dilution of primary antibody

  • Perform a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:3000) to determine optimal signal-to-noise ratio

  • Secondary antibody selection should match the host species (anti-rabbit for most OR5M1 antibodies)

• Blocking and washing:

  • Use 5% non-fat milk or BSA in TBST for blocking

  • Include extended washing steps (at least 3 × 10 minutes) to reduce background

• Positive controls:

  • Validated cell lines include Jurkat, HeLa, HepG2, and MDA-MB453, which have demonstrated detectable OR5M1 expression

  • Consider using recombinant OR5M1 protein as a positive control where available

• Specificity validation:

  • Include a peptide competition assay using the immunizing peptide to confirm specificity

  • Consider cross-reactivity with OR5M10 if using dual-specificity antibodies

What methodological considerations are important for immunofluorescence experiments using OR5M1 antibodies?

For successful immunofluorescence experiments with OR5M1 antibodies, researchers should consider the following methodological approaches:

• Cell fixation and permeabilization:

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

  • Permeabilize with 0.1-0.3% Triton X-100 for 5-10 minutes for optimal antibody access to this membrane protein

  • Consider mild fixation protocols that preserve membrane protein epitopes

• Blocking conditions:

  • Use 2-5% normal serum (from the species of secondary antibody) or BSA

  • Include 0.1% Tween-20 to reduce non-specific binding

  • Block for at least 1 hour at room temperature

• Antibody incubation:

  • Dilute primary antibody in the range of 1:100-1:500 in blocking buffer

  • Incubate overnight at 4°C for optimal binding

  • Use fluorophore-conjugated anti-rabbit secondary antibodies at 1:1000-1:2000

• Controls and validation:

  • Include a peptide competition control by pre-incubating the antibody with the immunizing peptide

  • Use cell lines with confirmed OR5M1 expression (e.g., MCF-7) as positive controls

  • Include a negative control omitting primary antibody to assess background fluorescence

• Image acquisition:

  • Expected subcellular localization is primarily cell membrane, with possible detection in ER/Golgi during protein processing

  • Optimize exposure settings to avoid saturation while maintaining signal visibility

How can researchers validate OR5M1 antibody specificity for their experiments?

Rigorous validation of antibody specificity is crucial for meaningful research outcomes. For OR5M1 antibodies, implement these validation approaches:

• Peptide competition assay:

  • Pre-incubate the antibody with 5-10× molar excess of immunizing peptide

  • Run parallel experiments with blocked and unblocked antibody

  • Specific signals should disappear in the peptide-blocked condition

• Knockout/knockdown validation:

  • Use siRNA or CRISPR-Cas9 to reduce OR5M1 expression

  • Compare signal between untreated and OR5M1-depleted samples

  • Specific signals should be reduced proportionally to knockdown efficiency

• Recombinant protein controls:

  • Test the antibody against recombinant OR5M1 protein in Western blot

  • Use OR5M1 protein with known tags (e.g., His-tag) to confirm detection

• Cross-reactivity assessment:

  • Test the antibody in cell lines known to express OR5M10 but not OR5M1

  • This is particularly important for antibodies targeting regions with sequence similarity between OR5M1 and OR5M10

  • Consider potential cross-reactivity with other olfactory receptors of the same family

• Multiple antibody validation:

  • Compare results using antibodies targeting different epitopes of OR5M1

  • Concordant results across antibodies increase confidence in specificity

What are the known challenges in detecting OR5M1 protein in experimental systems?

Researchers face several challenges when detecting OR5M1:

• Low expression levels:

  • Olfactory receptors often show tissue-specific and low-level expression

  • Consider using more sensitive detection methods or signal amplification approaches

• Membrane protein solubilization:

  • As a multi-pass membrane protein, OR5M1 may require specialized detergents for effective extraction

  • Optimization of lysis conditions may be necessary to prevent protein aggregation or epitope masking

• Cross-reactivity concerns:

  • High sequence similarity between olfactory receptor family members can lead to cross-reactivity

  • Even manufacturers acknowledge potential cross-reactivity with analogs: "Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between mouse CLU and all the analogs, therefore, cross reaction may still exist"

  • This statement, though specific to CLU in the source, reflects a common challenge with antibodies targeting protein families

• Post-translational modifications:

  • G-protein coupled receptors like OR5M1 often undergo phosphorylation and glycosylation

  • These modifications may affect antibody recognition depending on the epitope location

• Non-specific binding:

  • High background in immunoassays may complicate interpretation

  • Implementing stringent blocking and washing conditions is essential

How can researchers interpret contradictory results from different OR5M1 antibodies?

When facing contradictory results from different OR5M1 antibodies, researchers should systematically investigate the following factors:

• Epitope differences and accessibility:

  • Different antibodies target distinct regions (C-terminal, internal domains)

  • Certain epitopes may be masked depending on protein conformation or interaction partners

  • Compare the specific amino acid sequences targeted by each antibody

• Post-translational modifications:

  • Modifications near the epitope may affect antibody binding

  • Different antibodies may have varying sensitivities to phosphorylated, glycosylated, or otherwise modified OR5M1

• Antibody quality and batch variation:

  • Polyclonal antibodies can show batch-to-batch variation

  • Validate each new lot of antibody before use in critical experiments

• Experimental conditions:

  • Different antibodies may perform optimally under different conditions

  • Systematically compare fixation methods, incubation times, and buffer compositions

• Cross-reactivity analysis:

  • Assess whether differences may be due to detection of related proteins

  • Perform peptide competition assays with specific peptides for both OR5M1 and OR5M10

This approach aligns with established principles in antibody validation, as seen in other fields. For example, in research on viral antibodies, "the antibody response was not selectively reduced against individual epitopes but against all epitopes tested" , highlighting the importance of multi-epitope analysis.

How can OR5M1 antibodies be applied in functional studies of olfactory signaling?

OR5M1 antibodies can facilitate several approaches to studying olfactory signaling mechanisms:

• Receptor trafficking and localization:

  • Immunofluorescence microscopy to track OR5M1 localization under various stimuli

  • Co-localization studies with trafficking markers to understand receptor internalization and recycling

  • Live-cell imaging using antibody fragments to track receptor dynamics

• Protein-protein interaction studies:

  • Co-immunoprecipitation to identify OR5M1 interaction partners

  • Proximity ligation assays to visualize protein interactions in situ

  • Antibody-based pull-down coupled with mass spectrometry to identify the OR5M1 interactome

• Functional modulation studies:

  • Function-blocking antibodies to inhibit specific domains of OR5M1

  • Correlation of receptor phosphorylation state with signaling outcomes using phospho-specific antibodies

  • Analysis of ligand-induced conformational changes using conformation-specific antibodies

• Tissue distribution analysis:

  • Immunohistochemistry to map OR5M1 expression across olfactory and non-olfactory tissues

  • Single-cell analysis of OR5M1 expression in heterogeneous cell populations

These approaches can provide insights similar to those gained in other fields through antibody-based research, such as understanding "how the virus spreads within communities and which groups of people were most susceptible to being infected" , but applied to olfactory receptor biology.

What technical innovations might improve OR5M1 antibody specificity and utility?

Several emerging technologies and approaches could enhance OR5M1 antibody research:

• Single-chain variable fragment (scFv) development:

  • Generation of smaller antibody fragments with potentially better tissue penetration

  • Enhanced access to conformational epitopes that may be inaccessible to full IgG molecules

• Nanobody development:

  • Single-domain antibodies derived from camelid species

  • Superior recognition of conformational epitopes in membrane proteins

  • Potential for improved specificity between closely related olfactory receptors

• CRISPR-based epitope tagging:

  • Endogenous tagging of OR5M1 to enable detection with highly specific anti-tag antibodies

  • Circumvents issues of antibody cross-reactivity with related olfactory receptors

• Recombinant antibody engineering:

  • Affinity maturation to improve sensitivity

  • Engineering specificity to distinguish between OR5M1 and OR5M10

  • Development of bispecific antibodies for complex detection strategies

• Phospho-specific and conformation-specific antibodies:

  • Development of antibodies that recognize specific activated states of OR5M1

  • Tools to study receptor dynamics during signal transduction

These innovations follow principles similar to those seen in other fields, where technological advances have enabled "antibody tests [that] can provide public health officials with information on how many people in the community have been infected" , but applied specifically to advance OR5M1 research.

What best practices should researchers follow when designing experiments with OR5M1 antibodies?

Based on the compiled data, researchers should adhere to these best practices:

• Rigorous validation before experimental use:

  • Validate each antibody lot in systems with known OR5M1 expression

  • Include appropriate positive and negative controls

  • Perform peptide competition assays to confirm specificity

• Careful experimental design:

  • Select antibodies based on the specific research question and required applications

  • Consider using multiple antibodies targeting different epitopes when possible

  • Optimize protocols specifically for OR5M1 detection rather than using generic protocols

• Transparent reporting:

  • Document complete antibody information (catalog number, lot, dilution)

  • Report detailed methodological conditions

  • Acknowledge limitations and potential cross-reactivity

• Cell and tissue selection:

  • Verify OR5M1 expression in selected experimental systems

  • Consider using validated cell lines (MCF-7, HeLa, HepG2, Jurkat) as positive controls

• Integration with other methods:

  • Combine antibody-based detection with orthogonal methods (RT-PCR, mass spectrometry)

  • Use genetic manipulation to validate antibody specificity

Following these guidelines will enhance experimental reproducibility and data reliability in OR5M1 research, aligning with broader principles in antibody-based research where "understanding a test's accuracy [requires] scientists look at sensitivity and specificity" .

What future directions should OR5M1 antibody development take to address current limitations?

Future development of OR5M1 antibodies should focus on:

• Increased specificity:

  • Development of monoclonal antibodies with enhanced specificity for OR5M1 over OR5M10

  • Targeting of unique epitopes that differentiate between closely related olfactory receptors

  • Rigorous cross-reactivity testing across the olfactory receptor family

• Functional antibodies:

  • Development of antibodies that can modulate OR5M1 function

  • Conformation-specific antibodies that recognize active vs. inactive states

  • Phospho-specific antibodies to study receptor activation dynamics

• Advanced detection capabilities:

  • Directly conjugated antibodies to eliminate secondary antibody requirements

  • Super-resolution microscopy-compatible fluorophore conjugates

  • Antibodies optimized for live-cell imaging applications

• Expanded validation standards:

  • Standardized validation across multiple cell types and tissues

  • Genetic knockout validation in relevant model systems

  • Cross-validation with orthogonal detection methods