OR5M9 Antibody

What is OR5M9 and why is it studied in molecular research?

OR5M9 (Olfactory Receptor Family 5 Subfamily M Member 9) is a member of the olfactory receptor protein family. These receptors interact with odorant molecules in the nose to initiate neuronal responses that trigger smell perception. OR5M9 belongs to the large family of G-protein-coupled receptors (GPCRs) with a characteristic 7-transmembrane domain structure . Also known as OR11-190, this receptor is encoded by a single coding-exon gene .

The study of OR5M9 is significant because:

• It contributes to understanding the molecular basis of olfaction

• It represents one of the largest gene families in the human genome

• It shares structural similarities with many neurotransmitter and hormone receptors

• It may have potential roles beyond olfaction that remain to be discovered

What applications are OR5M9 antibodies most commonly validated for?

Based on comprehensive product information across multiple suppliers, OR5M9 antibodies have been validated for several key applications:

Multiple antibody sources show consistent validation for these applications, with Western blotting and ELISA being the most thoroughly validated methods .

What are the recommended storage conditions for maintaining OR5M9 antibody activity?

For optimal preservation of OR5M9 antibody activity, follow these evidence-based storage protocols:

• Long-term storage: Store at -20°C for up to one year

• Short-term storage: For frequent use, store at 4°C for up to one month

• Buffer composition: Most commercial OR5M9 antibodies are supplied in PBS with 50% glycerol and 0.02% sodium azide at pH 7.4

• Avoid repeated freeze-thaw cycles as this can lead to protein denaturation and loss of antibody activity

• Aliquoting: For antibodies that will be used multiple times, create small working aliquots to minimize freeze-thaw cycles

The liquid formulation with glycerol helps prevent freezing damage and maintains antibody stability during storage.

How can researchers validate the specificity of OR5M9 antibodies in experimental systems?

Validating OR5M9 antibody specificity is critical for generating reliable research data. Multiple approaches should be implemented:

• Peptide competition assays: Pre-incubate the OR5M9 antibody with its immunizing peptide. Complete signal abolishment in subsequent assays indicates specificity . The validation data from one antibody provider shows successful peptide blocking in immunofluorescence assays with MCF7 cells .

• Positive control tissues/cells: Validated positive controls for OR5M9 detection include:

  • Jurkat cells (human T lymphocyte line)

  • HepG2 cells (human liver cancer line)

  • HeLa cells (human cervical cancer line)

  • MCF7 cells (human breast cancer line)

• Genetic approaches:

  • Use OR5M9 knockout/knockdown systems as negative controls

  • Test antibody reactivity in overexpression systems

  • Compare staining patterns across multiple antibodies targeting different OR5M9 epitopes

• Cross-reactivity assessment: Evaluate antibody performance across species. Several OR5M9 antibodies show cross-reactivity with human and mouse OR5M9, while some have broader reactivity including bovine, canine, guinea pig, equine, porcine, rabbit and rat samples .

• Immunoblot profile analysis: Verify that the detected molecular weight matches the predicted size of OR5M9 (approximately 35 kDa) .

What are the most effective strategies for troubleshooting inconsistent OR5M9 antibody results in immunoassays?

When facing inconsistent results with OR5M9 antibodies, implement this systematic troubleshooting approach:

• Antibody validation:

  • Confirm antibody specificity using techniques described in Question 4

  • Verify antibody reactivity using positive control lysates from Jurkat, HepG2, or HeLa cells

• Sample preparation optimization:

  • For Western blot: Ensure complete protein denaturation with appropriate buffers

  • For IF/ICC: Compare different fixation methods (paraformaldehyde vs. methanol) as fixation can affect epitope accessibility

  • For ELISA: Test different coating concentrations and blocking agents

• Protocol adjustment:

  • Optimize antibody concentration (test dilutions ranging from 1:100-1:3000 depending on application)

  • Extend primary antibody incubation time (overnight at 4°C vs. 1-2 hours at room temperature)

  • Test various antigen retrieval methods for tissue sections

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time or concentration to reduce background

• Detection system evaluation:

  • Compare different secondary antibodies and detection methods

  • For low-abundance targets, consider signal amplification methods

• Examine experimental variables:

  • Account for biological variation in OR5M9 expression across different cell types and tissues

  • Consider potential post-translational modifications that might affect antibody binding

What considerations are important when designing co-immunoprecipitation experiments to identify OR5M9 binding partners?

Co-immunoprecipitation (Co-IP) of OR5M9 requires special considerations due to its transmembrane nature:

• Antibody selection:

  • Use antibodies raised against internal regions or C-terminus of OR5M9

  • Validate that the antibody can recognize native (non-denatured) OR5M9

• Membrane protein solubilization:

  • Use mild detergents (e.g., CHAPS, digitonin, or NP-40) to solubilize OR5M9 without disrupting protein-protein interactions

  • Optimize detergent concentration and extraction time

  • Include protease inhibitors to prevent degradation

• Crosslinking considerations:

  • Consider using membrane-permeable crosslinkers to stabilize transient interactions

  • Optimize crosslinking conditions to capture physiologically relevant interactions

• Control experiments:

  • Include IgG control from the same species as the OR5M9 antibody

  • Include lysates from cells not expressing OR5M9 as negative controls

  • Consider using tagged OR5M9 constructs as alternative precipitation targets

• Validation of interactions:

  • Confirm interactions by reverse Co-IP when possible

  • Use proximity ligation assays to validate interactions in situ

  • Consider mass spectrometry to identify novel binding partners

• Data analysis:

  • Account for common contaminants in Co-IP experiments

  • Focus on enriched proteins compared to control samples

  • Consider the biological relevance of identified interactions in olfactory signaling

How should researchers interpret conflicting results between different OR5M9 antibody clones?

When faced with discrepancies between different OR5M9 antibody clones, consider these methodological approaches:

• Epitope mapping analysis:

  • Compare the immunogen sequences of different antibody clones

  • Many OR5M9 antibodies target different internal regions:

    • Amino acids 197-246 (middle region)

    • Amino acids 257-290 (C-terminal region)

    • Amino acids 204-254 (internal region)

  • Different epitopes may be differentially accessible depending on experimental conditions

• Post-translational modification considerations:

  • Determine if discrepant antibodies might recognize different post-translationally modified forms of OR5M9

  • Consider if certain antibodies might be sensitive to phosphorylation or glycosylation states

• Reconciliation strategies:

  • Use multiple antibodies targeting different epitopes as cross-validation

  • Employ orthogonal techniques (e.g., mass spectrometry) to confirm protein identity

  • Consider epitope tagging approaches to validate antibody specificity

• Cross-reactivity assessment:

  • Evaluate whether discrepancies might arise from cross-reactivity with closely related olfactory receptors

  • Perform specificity tests using overexpression and knockdown systems

• Contextual interpretation:

  • Consider that discrepancies might reflect biological reality rather than technical artifacts

  • Document experimental conditions thoroughly to allow for accurate comparison

What experimental approaches are most effective for studying OR5M9 in olfactory signaling pathways?

To effectively study OR5M9 in olfactory signaling contexts, consider these methodological approaches:

• Heterologous expression systems:

  • Express OR5M9 in cell lines (HEK293, Hana3A) engineered to support olfactory receptor trafficking and signaling

  • Couple with calcium imaging or cAMP assays to measure functional responses to potential ligands

• CRISPR-based genome editing:

  • Generate OR5M9 knockout models to assess loss-of-function phenotypes

  • Create knock-in reporter systems to monitor endogenous OR5M9 expression

• Functional assays:

  • Implement high-throughput screening of odorant libraries to identify OR5M9 ligands

  • Use bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) assays to monitor OR5M9 activation and G-protein coupling

• Structural biology approaches:

  • Apply computational modeling based on GPCR structures to predict OR5M9 ligand binding sites

  • Consider nanobody development approaches as described in recent SARS-CoV-2 research, which could be adapted for OR5M9 structural studies

• Single-cell analyses:

  • Implement single-cell RNA sequencing to characterize cells expressing OR5M9

  • Use spatial transcriptomics to map OR5M9 expression in olfactory tissues

• Antibody applications:

  • Use validated OR5M9 antibodies for immunohistochemistry to map receptor distribution in olfactory epithelia

  • Employ proximity ligation assays to identify in situ protein-protein interactions

How can researchers optimize detection of OR5M9 in tissues with low expression levels?

Detecting low-abundance OR5M9 requires specialized approaches:

• Sample enrichment strategies:

  • Implement subcellular fractionation to concentrate membrane proteins

  • Consider immunoprecipitation before Western blotting to enrich OR5M9

  • Use optimized extraction buffers specifically designed for transmembrane proteins

• Signal amplification methods:

  • For immunohistochemistry/immunofluorescence:

    • Utilize tyramide signal amplification (TSA)

    • Consider rolling circle amplification

    • Use highly sensitive detection systems (e.g., Quantum dots)

  • For Western blot:

    • Use high-sensitivity chemiluminescent substrates

    • Consider fluorescent secondary antibodies with near-infrared detection systems

• Optimized antibody protocols:

  • Extend primary antibody incubation times (overnight at 4°C)

  • Optimize antibody concentrations through careful titration

  • Reduce washing stringency to preserve low-level signals

• Alternative detection methods:

  • Consider RNAscope or BaseScope for sensitive mRNA detection

  • Use highly sensitive mass spectrometry approaches for protein detection

  • Implement digital PCR for quantification of OR5M9 transcripts

• Biological amplification:

  • Consider using cell systems with inducible OR5M9 overexpression

  • Implement CRISPR activation (CRISPRa) to upregulate endogenous OR5M9

What are the critical considerations for designing experiments to study post-translational modifications of OR5M9?

Investigating post-translational modifications (PTMs) of OR5M9 requires specialized experimental design:

• Modification-specific antibodies:

  • Consider developing or sourcing antibodies that specifically recognize phosphorylated, glycosylated, or ubiquitinated forms of OR5M9

  • Validate specificity using dephosphorylation, deglycosylation, or deubiquitination treatments

• Enrichment strategies:

  • For phosphorylation: Use phospho-protein/peptide enrichment techniques (IMAC, TiO₂)

  • For glycosylation: Implement lectin affinity chromatography

  • For ubiquitination: Use tandem ubiquitin binding entities (TUBEs)

• Mass spectrometry approaches:

  • Develop targeted MS methods for specific OR5M9 peptides and their modified forms

  • Consider top-down proteomics to preserve intact protein and all modifications

  • Implement electron transfer dissociation (ETD) or electron capture dissociation (ECD) fragmentation to preserve labile modifications

• Site-directed mutagenesis:

  • Identify potential modification sites through computational prediction

  • Generate site-specific mutants (e.g., S/T→A for phosphorylation, K→R for ubiquitination)

  • Assess functional consequences of preventing specific modifications

• Temporal dynamics:

  • Design time-course experiments to capture dynamic changes in PTMs following receptor activation

  • Consider pulse-chase approaches to track PTM turnover

• Pharmacological modulators:

  • Use kinase inhibitors, deubiquitinase inhibitors, or glycosylation inhibitors to manipulate PTM states

  • Monitor effects on receptor trafficking, signaling, and turnover

What are the most reliable positive and negative controls for OR5M9 antibody validation experiments?

Implementing appropriate controls is essential for rigorous OR5M9 antibody validation:

Positive Controls:

• Cell lines with confirmed OR5M9 expression:

  • Jurkat cells (human T lymphocyte line)

  • HepG2 cells (human liver cancer line)

  • HeLa cells (human cervical cancer line)

  • MCF7 cells (human breast cancer line)

• Tissue samples:

  • Human nasal epithelium (physiological expression site)

  • Other tissues with reported OR5M9 expression

• Recombinant systems:

  • Cells transiently transfected with OR5M9 expression constructs

  • Purified recombinant OR5M9 protein (available from sources like ABIN7550464)

Negative Controls:

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide to block specific binding

  • Multiple vendors offer blocking peptides specific to their antibodies

• Genetic approaches:

  • OR5M9 knockdown/knockout cell lines

  • Tissues from OR5M9-deficient models

• Antibody controls:

  • Isotype-matched irrelevant antibodies (rabbit IgG for most OR5M9 antibodies)

  • Primary antibody omission controls

  • Secondary antibody-only controls

• Application-specific controls:

  • For Western blot: Loading controls and molecular weight markers

  • For ICC/IF: Counterstains to verify cellular morphology and localization

  • For ELISA: Background subtraction controls

What specialized techniques can be employed to study OR5M9 protein-ligand interactions?

Investigating OR5M9 ligand interactions requires specialized techniques that account for its GPCR nature:

• Cell-based functional assays:

  • Calcium imaging: Monitor intracellular calcium flux following receptor activation

  • cAMP assays: Measure changes in cAMP levels using FRET or BRET-based sensors

  • β-arrestin recruitment: Assess receptor activation through monitoring β-arrestin recruitment

• Binding assays:

  • Radioligand binding: Develop radiolabeled ligands for OR5M9

  • Time-resolved fluorescence resonance energy transfer (TR-FRET): Use lanthanide-labeled ligands

  • Surface plasmon resonance (SPR): Assess binding kinetics with purified receptor components

• Structural approaches:

  • Computational modeling: Apply techniques from recent advances in protein design

  • Molecular dynamics simulations: Predict binding pockets and ligand interactions

  • Cross-linking mass spectrometry: Identify residues involved in ligand binding

• High-throughput screening platforms:

  • Luciferase reporter systems coupled to OR5M9 activation

  • Automated calcium imaging for odorant library screening

  • Cell microarray platforms for parallel testing of multiple conditions

• Advanced microscopy approaches:

  • Single-molecule tracking to monitor receptor dynamics

  • Super-resolution microscopy to visualize receptor clustering

  • FRET microscopy to detect conformational changes upon ligand binding

• AI-assisted techniques:

  • Apply active learning approaches similar to those used in antibody-antigen binding prediction

  • Implement virtual screening of potential OR5M9 ligands

How can researchers implement machine learning approaches to improve OR5M9 antibody selection and experimental design?

Machine learning offers powerful tools for optimizing OR5M9 research:

• Antibody selection optimization:

  • Implement active learning strategies similar to those used for antibody-antigen binding prediction

  • Use computational models to predict epitope accessibility in different experimental conditions

  • Develop tools to assess the likelihood of cross-reactivity with related olfactory receptors

• Experimental design enhancement:

  • Apply sequential experimental design algorithms to determine optimal antibody dilutions and incubation conditions

  • Implement transfer learning from well-characterized antibodies to new OR5M9 antibodies

  • Use Bayesian optimization to efficiently search the parameter space of experimental conditions

• Data analysis augmentation:

  • Train image analysis algorithms to quantify immunofluorescence staining patterns

  • Develop models for automated Western blot band quantification

  • Implement anomaly detection to identify experimental artifacts

• Epitope prediction:

  • Use sequence-based and structure-based models to identify optimal epitopes for antibody generation

  • Apply algorithms similar to those used in the Virtual Lab for nanobody design

  • Implement conformational epitope prediction algorithms

• Cross-reactivity prediction:

  • Develop models to predict potential cross-reactivity with other olfactory receptors

  • Integrate proteome-wide epitope mapping to identify potential off-target binding

• Integration with experimental data:

  • Implement lab-in-the-loop approaches as described for antibody-antigen binding prediction

  • Use active learning to select the most informative experiments to perform next

  • Incorporate real-time experimental feedback to refine predictions

What novel approaches from related research fields could be applied to OR5M9 antibody development and application?

Several cutting-edge approaches from related fields could advance OR5M9 research:

• Nanobody development technologies:

  • Adapt computational pipelines from SARS-CoV-2 nanobody design

  • Implement the Virtual Lab approach combining ESM, AlphaFold-Multimer, and Rosetta for OR5M9-specific binders

  • Create single-domain antibodies with enhanced access to GPCR binding pockets

• Immune modelling systems:

  • Utilize immune modeling platforms for rational OR5M9 antibody design

  • Implement in vitro human platforms for co-cultures to test antibody specificity

  • Design experimental models specifically for transmembrane protein targets

• Structure-based design approaches:

  • Apply cryo-EM techniques optimized for membrane proteins

  • Use computational structure prediction tools (AlphaFold) to model OR5M9 structure

  • Design antibodies targeting specific conformational states of OR5M9

• Single-cell antibody platforms:

  • Implement massively parallel antibody discovery platforms

  • Use droplet microfluidics for single-cell screening of OR5M9 antibody candidates

  • Apply phage display technologies with customized selection conditions for GPCRs

• Advanced imaging technologies:

  • Adapt expansion microscopy for enhanced visualization of OR5M9 in tissues

  • Implement DNA-PAINT for super-resolution imaging of OR5M9 distribution

  • Use correlative light and electron microscopy to map OR5M9 at the nanoscale

• AI-driven experimental design:

  • Implement virtual screening of antibody libraries against predicted OR5M9 structures

  • Use generative models to design novel OR5M9-specific peptide immunogens

  • Apply evolutionary algorithms to optimize antibody affinity and specificity