OR5T3 Antibody

What is OR5T3 protein and what is its biological significance?

OR5T3 (Olfactory receptor 5T3) is a member of the G-protein coupled receptor 1 family functioning as an olfactory receptor involved in the detection of odorant molecules in the olfactory sensory neurons. It is encoded by the OR5T3 gene (ID: 390154) in humans with UniProt ID Q8NGG3 . The protein has a calculated molecular weight of approximately 38 kDa, though observed molecular weight can vary due to post-translational modifications . OR5T3 consists of 320 amino acids forming the characteristic seven-transmembrane domain structure typical of G-protein coupled receptors. The full amino acid sequence reveals the structural organization necessary for its function in olfactory signal transduction .

Research interest in OR5T3 stems primarily from its role in olfactory perception, but recent studies have also investigated potential ectopic expression in non-olfactory tissues. The protein functions by binding odorant molecules and initiating a signaling cascade that ultimately results in the perception of specific odors. Understanding its structure, expression patterns, and function contributes to our knowledge of the molecular basis of olfaction.

What are the characteristics of available OR5T3 antibodies?

Based on technical data from multiple antibody manufacturers, researchers have several options for OR5T3 antibodies with varying characteristics:

All currently available commercial antibodies against OR5T3 are rabbit polyclonal antibodies . They are generated by immunizing rabbits with synthetic peptides derived from human OR5T3 sequences. The antibodies are typically purified by affinity chromatography against the immunizing peptide, which enhances their specificity for the target protein . Cross-reactivity potential is determined by sequence homology between species, explaining the observed reactivity patterns across multiple species.

What applications are OR5T3 antibodies validated for?

Current validation data for OR5T3 antibodies covers multiple applications with specific recommended dilutions:

For Western blot applications, OR5T3 antibodies detect bands at the calculated molecular weight of approximately 38 kDa, although variations may be observed due to post-translational modifications or alternative splicing . Immunofluorescence applications have been validated in several cell lines, with MCF7 cells specifically noted as a positive control . The optimal dilutions should be determined by the end user through careful titration experiments, as the ideal concentration may vary depending on the specific experimental conditions and sample types .

How should OR5T3 antibodies be stored to maintain optimal activity?

Proper storage conditions are essential for maintaining antibody functionality:

Commercially available OR5T3 antibodies are typically supplied in phosphate buffered saline (pH 7.4) containing 50% glycerol, 0.02% sodium azide, and sometimes additional stabilizers like 0.5% bovine serum albumin . Under these conditions, OR5T3 antibodies maintain activity for approximately 12 months from the date of receipt when stored properly at -20°C . For frequent use over a short period, storage at 4°C for up to one month is acceptable, but repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of antibody activity .

How can researchers optimize immunostaining protocols for low-abundance OR5T3 detection?

Detecting low-abundance OR5T3 expression requires careful protocol optimization across multiple parameters:

• Signal Enhancement Strategies:

  • Tyramide signal amplification (TSA) can increase sensitivity 10-100 fold by enzymatically depositing additional fluorophores at the site of antibody binding

  • Quantum dot conjugated secondary antibodies provide superior photostability and brighter signals for detecting minimal expression

  • Poly-HRP detection systems offer amplified signal with low background through increased enzyme density per binding event

• Sample Preparation Optimization:

  • Extended fixation (15-20 minutes in 4% paraformaldehyde) improves epitope retention without compromising accessibility

  • Antigen retrieval in citrate buffer pH 6.0 at 95°C increases epitope accessibility, particularly important for formalin-fixed tissues

  • Blocking with 5% BSA + 5% normal serum reduces non-specific binding while maintaining specific signal intensity

• Antibody Incubation Parameters:

  • Primary antibody incubation at 1:100 dilution overnight at 4°C maximizes binding kinetics and equilibrium

  • Including 0.1% Triton X-100 in antibody diluent enhances penetration, particularly for membrane proteins like OR5T3

  • Secondary antibody at 1:200 for 2 hours provides optimal signal-to-noise ratio

When validating immunofluorescence results, peptide competition controls are essential. For example, immunofluorescence analysis of MCF7 cells demonstrated positive OR5T3 staining that was effectively blocked when the antibody was pre-incubated with the synthesized peptide, confirming specificity of the observed signal .

What are the most effective validation strategies for OR5T3 antibody specificity?

Rigorous validation of OR5T3 antibody specificity requires multiple complementary control strategies:

Peptide competition assays are particularly valuable for polyclonal antibodies like those available for OR5T3. By pre-incubating the antibody with the immunizing peptide, specific binding sites are blocked, and any remaining signal likely represents non-specific binding. This approach has been successfully employed with OR5T3 antibodies, where pre-incubation with the synthesized peptide blocked immunofluorescence staining in MCF7 cells .

For western blot applications, additional controls should include samples from tissues or cell lines with known OR5T3 expression levels, as well as molecular weight markers to confirm the expected band size of approximately 38 kDa. Any unexpected bands should be carefully investigated to determine if they represent alternatively spliced variants, post-translational modifications, or non-specific binding.

How does the choice of epitope affect OR5T3 antibody performance?

The location of the target epitope significantly impacts OR5T3 antibody performance across different applications:

• Transmembrane Domain Considerations:

  • Antibodies targeting extracellular domains work well in non-permeabilized immunofluorescence applications

  • Antibodies against intracellular loops perform better in Western blot after complete denaturation

  • C-terminal epitopes are often more accessible in fixed tissues but may be affected by protein-protein interactions

• Application-Specific Effects:

  • For Western blot, antibodies recognizing linear epitopes outperform those targeting conformational epitopes

  • For IHC/IF, antibodies against conformational epitopes may better recognize the native protein structure

  • Immunoprecipitation applications typically require antibodies recognizing surface-exposed epitopes in the protein's native state

• OR5T3 Structural Implications:

  • As a seven-transmembrane G-protein coupled receptor, OR5T3 presents unique challenges for antibody recognition

  • The N-terminal domain is extracellular and relatively accessible in intact cells

  • The C-terminal domain is intracellular and may be involved in signaling complex formation

Currently available commercial antibodies target different regions of the OR5T3 protein. For example, the Boster Bio antibody (A17573) targets a peptide derived from amino acids 86-135 , which encompasses parts of the transmembrane domains and extracellular loops. This region appears to provide good accessibility and specificity across multiple applications.

What are the challenges in interpreting OR5T3 immunostaining in different tissue contexts?

Interpreting OR5T3 immunostaining in different tissue contexts requires consideration of several confounding factors:

• Non-specific Binding Concerns:

  • Necrotic tissue areas show increased non-specific antibody retention due to exposed hydrophobic regions

  • Highly vascularized regions may show false-positive staining due to endogenous peroxidase activity if using HRP-based detection

  • Lipid-rich tissues can trap hydrophobic antibody regions, necessitating more stringent washing protocols

• Expression Context Considerations:

  • OR5T3 is primarily expressed in olfactory sensory neurons, so detection in other tissues requires careful validation

  • Ectopic expression may reflect dysregulation rather than functional relevance

  • Inflammatory conditions can induce transient expression of multiple olfactory receptors as part of stress response

• Technical Artifacts:

  • Edge effects in tissue sections can be misinterpreted as positive staining due to reagent trapping

  • Antigen retrieval can unmask epitopes not normally accessible in vivo, potentially leading to false positives

  • Fixation gradient in larger specimens creates variable staining patterns that may not reflect true expression differences

Because OR5T3 belongs to a large family of olfactory receptors with sequence similarities, cross-reactivity is a significant concern. When examining tissues not typically associated with OR5T3 expression, researchers should implement multiple detection methods (IHC, IF, in situ hybridization) and correlate results with mRNA expression data to minimize misinterpretation.

What is the optimal western blotting protocol for OR5T3 detection?

For robust Western blot detection of OR5T3, the following protocol incorporates critical optimizations for this membrane protein:

• Sample Preparation:

  • Lyse cells in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0) containing protease inhibitor cocktail

  • Sonicate briefly (3 × 10s pulses) to shear DNA and reduce viscosity

  • Centrifuge at 14,000 × g for 15 minutes to clear debris

  • Add 4× Laemmli buffer with 100 mM DTT and heat at 95°C for 5 minutes to ensure complete denaturation

• Gel Electrophoresis and Transfer:

  • Load 50-75 μg total protein per lane on 10% SDS-PAGE

  • Run at 100V until dye front reaches bottom

  • Transfer to PVDF membrane (preferred over nitrocellulose for hydrophobic proteins) at 100V for 90 minutes in cold transfer buffer containing 20% methanol

• Immunodetection:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with OR5T3 antibody at 1:1000-3000 dilution in 5% BSA/TBST overnight at 4°C

  • Wash 4 × 5 minutes with TBST

  • Incubate with HRP-conjugated secondary antibody at 1:5000 for 1 hour at room temperature

  • Wash 4 × 5 minutes with TBST

  • Develop using enhanced chemiluminescence substrate

• Expected Results:

  • Primary band at approximately 38 kDa (calculated molecular weight)

  • Possible additional bands may represent post-translational modifications or protein complexes

  • Verification with positive control is essential for interpretation

This protocol has been optimized to minimize background while maximizing specific signal for OR5T3 detection. The key factors influencing successful detection include complete denaturation of the sample, sufficient protein loading, and extended primary antibody incubation at the appropriate dilution.

How can researchers quantitatively analyze OR5T3 expression across experimental conditions?

Quantitative analysis of OR5T3 requires standardized methods that account for technical and biological variability:

• Immunohistochemistry/Immunofluorescence Quantification:

  • Use consistent tissue processing, staining, and imaging parameters across all samples

  • Apply H-score methodology (staining intensity × percentage of positive cells) for semi-quantitative analysis

  • Utilize automated image analysis software with validated algorithms to remove subjective interpretation

  • Include calibration standards on each slide for normalization between experiments

  • Analyze multiple fields per sample to account for expression heterogeneity

• Western Blot Quantification:

  • Include concentration gradient of recombinant standard or positive control lysate on each blot

  • Normalize to multiple housekeeping proteins appropriate for the tissue or cell type being studied

  • Use fluorescent secondary antibodies for wider linear detection range compared to chemiluminescence

  • Perform technical replicates (minimum of three) and biological replicates for statistical validation

  • Apply appropriate statistical tests based on data distribution

• qPCR Correlation:

  • Design primers spanning exon-exon junctions to eliminate genomic DNA amplification

  • Validate primer efficiency (should be 90-110%) with standard curves

  • Use minimum of three reference genes validated for stability in the specific experimental conditions

  • Apply ΔΔCt method with appropriate statistical analysis

When comparing OR5T3 expression across different conditions, it is essential to maintain consistent protocols throughout the study. For immunofluorescence applications, antibody concentrations between 1:100-1:500 have been validated , but the optimal dilution should be determined empirically for each experimental system.

What approaches can resolve discrepancies between OR5T3 protein and mRNA expression data?

Resolving discrepancies between protein and mRNA data requires systematic investigation of both technical and biological factors:

• Technical Validation:

  • Confirm antibody specificity using peptide competition or genetic knockout/knockdown controls

  • Verify primer specificity through sequencing of amplicons and melt curve analysis

  • Test multiple antibodies targeting different epitopes of OR5T3 to rule out epitope-specific issues

  • Employ absolute quantification methods for both protein and mRNA to enable direct comparison

• Biological Explanations:

  • Post-transcriptional regulation (miRNA targeting, RNA stability) may cause discordance between mRNA and protein levels

  • Post-translational modifications can affect antibody recognition without altering mRNA levels

  • Protein turnover rates often differ significantly from mRNA degradation rates

  • Sub-cellular localization may impact detection efficiency, particularly for membrane proteins like OR5T3

• Reconciliation Approaches:

  • Temporal analysis to detect potential time lags between transcription and translation

  • Single-cell analysis to resolve potential cellular heterogeneity that might be masked in bulk tissue analysis

  • Polysome profiling to determine translational efficiency of OR5T3 mRNA

  • Proteasome inhibition studies to assess protein degradation rates

When investigating OR5T3 expression, it is particularly important to consider that as a G-protein coupled receptor, its trafficking to the cell membrane and internalization dynamics may significantly affect detection by different methods. Correlation between protein detection methods (like Western blot vs. immunofluorescence) and mRNA quantification should be interpreted with these factors in mind.

What is the recommended protocol for immunofluorescence detection of OR5T3?

For optimal immunofluorescence detection of OR5T3, the following detailed protocol incorporates critical parameters for membrane protein detection:

• Cell/Tissue Preparation:

  • For cells: Grow on sterile coverslips or chamber slides to 70-80% confluence

  • For tissues: Prepare frozen sections (8-10 μm) or paraffin sections with appropriate antigen retrieval

  • Wash twice with PBS (pH 7.4) to remove media components or mounting medium

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

  • Permeabilize with 0.1-0.2% Triton X-100 for 10 minutes to facilitate antibody access to intracellular epitopes

• Immunostaining:

  • Block with 5% normal serum (from same species as secondary antibody) in PBS for 1 hour at room temperature

  • Dilute OR5T3 antibody at 1:100-1:500 in antibody diluent (typically 1% BSA in PBS)

  • Incubate with primary antibody overnight at 4°C in a humidified chamber

  • Wash 3× with PBS, 5 minutes each

  • Incubate with fluorophore-conjugated secondary antibody (1:200-1:500) for 1 hour at room temperature

  • Wash 3× with PBS, 5 minutes each

  • Counterstain nuclei with DAPI (1 μg/mL) for 5 minutes

  • Mount with anti-fade mounting medium to preserve fluorescence

• Critical Controls:

  • Secondary antibody-only control to assess non-specific binding

  • Peptide competition control where available (pre-incubate antibody with immunizing peptide)

  • Positive control (e.g., MCF7 cells have shown detectable expression)

  • Negative control (tissue or cell line known not to express OR5T3)

• Imaging Parameters:

  • Capture images using consistent exposure settings across all samples

  • Include all appropriate controls in the same imaging session

  • Collect z-stacks for thick specimens to ensure complete signal capture

  • Process all images with identical parameters for valid comparison

This protocol has been demonstrated to provide specific staining of OR5T3 in multiple cell types, with successful validation using peptide competition controls . The specificity of the staining pattern should be carefully evaluated, with particular attention to the expected subcellular localization of OR5T3 as a membrane protein.