OR5T2 Antibody

What is OR5T2 and why is it significant in olfactory research?

OR5T2 (Olfactory receptor family 5, subfamily T, member 2), also known as OR11-177, is a 329 amino acid multi-pass membrane protein that functions as an odorant receptor. It belongs to the G protein-coupled receptor family that localizes to the cilia of olfactory sensory neurons where they bind to various odor molecules .

The significance of OR5T2 in olfactory research stems from its role in the signal transduction pathway that propagates nerve impulses throughout the body, ultimately leading to the perception of smell. OR5T2 is part of the largest gene family in the human genome - the olfactory receptor genes - making it an important target for understanding the molecular basis of olfaction .

What are the common applications for OR5T2 antibodies in laboratory research?

OR5T2 antibodies are primarily utilized in the following research applications:

These applications allow researchers to study OR5T2 expression, localization, and interactions in various experimental contexts .

How should OR5T2 antibodies be stored to maintain optimal activity?

Most commercial OR5T2 antibodies should be stored according to these guidelines:

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

• Short-term storage and frequent use: 4°C for up to one month

• Aliquoting is recommended to avoid repeated freeze/thaw cycles that can degrade antibody performance

• Most preparations contain glycerol (typically 50%) and preservatives like Proclin-300 (0.03%) or sodium azide in a buffer solution (often PBS, pH 7.4)

Before use, antibodies should be thawed completely and mixed gently to ensure homogeneity. Optimal antibody dilutions should be determined experimentally for each specific application .

What validation steps should be performed before using OR5T2 antibodies in critical experiments?

A comprehensive validation protocol for OR5T2 antibodies should include:

• Specificity testing:

  • Positive control: Use samples known to express OR5T2 (based on literature)

  • Negative control: Use samples known not to express OR5T2

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide to confirm specificity

• Application-specific validation:

  • For Western blot: Confirm correct molecular weight (~36 kDa) and single band specificity

  • For immunofluorescence: Compare staining pattern with known cellular localization (membrane-associated)

  • For ELISA: Establish standard curves with recombinant OR5T2 protein

• Cross-reactivity assessment:

  • Test against closely related olfactory receptors to ensure specificity

  • Examine multiple species if cross-species reactivity is claimed by the manufacturer

These validation steps are critical for confirming antibody performance before proceeding to experimental applications and should be documented in your research protocols .

What are the optimal dilution ranges for OR5T2 antibodies in different applications?

Based on the compiled data from multiple suppliers, the recommended dilution ranges for OR5T2 antibodies vary by application:

It's important to note that these represent starting points, and the optimal dilution should be determined empirically for each specific experiment. Factors affecting optimal dilution include:

• Sample type and target protein abundance

• Detection method (e.g., chemiluminescence vs. fluorescence for WB)

• Incubation conditions (time, temperature)

• Blocking reagents used

How can I troubleshoot weak or non-specific signals when using OR5T2 antibodies?

When encountering weak or non-specific signals with OR5T2 antibodies, the following methodological approach can help resolve common issues:

For weak signals:

• Increase antibody concentration or incubation time

• Optimize protein loading (for WB) or cell density (for IF/ICC)

• Enhance signal detection methods (e.g., more sensitive substrates for HRP)

• Ensure sample preparation preserves protein integrity (use protease inhibitors)

• Check expression levels in your experimental system as OR5T2 may have tissue-specific expression patterns

For non-specific signals:

• Increase blocking duration or change blocking reagent

• Reduce antibody concentration

• Increase wash steps duration and frequency

• Pre-adsorb antibody with non-specific proteins

• Optimize fixation methods for IF/ICC

• Verify antibody specificity with appropriate controls

Sample preparation considerations:

• For membrane proteins like OR5T2, ensure appropriate lysis buffers that effectively solubilize membrane proteins

• Consider using specialized membrane protein extraction kits

• Avoid excessive heat during sample preparation which can cause protein aggregation

How do OR5T2 antibodies perform across species, and what are the key considerations for cross-species experiments?

The cross-species reactivity of OR5T2 antibodies varies significantly based on epitope conservation and antibody generation methods:

Key considerations for cross-species applications:

• Epitope analysis: Compare the immunogen sequence used to generate the antibody with the corresponding region in your target species. Higher sequence identity increases likelihood of cross-reactivity.

• Positive control validation: Always include a positive control from the species of interest to confirm antibody performance.

• Optimization requirements: Cross-species applications typically require modified protocols:

  • Higher antibody concentrations may be needed

  • Longer incubation times often improve signal

  • Modified blocking conditions may reduce background

  • Species-specific secondary antibodies are critical

• Independent validation: Western blot validation in your species of interest is strongly recommended before proceeding to more complex applications .

What approaches can be used to distinguish between OR5T2 and other closely related olfactory receptors in complex samples?

Distinguishing OR5T2 from other closely related olfactory receptors requires specialized approaches due to structural and sequence similarities within this large receptor family:

• Epitope-specific antibodies: Use antibodies targeting unique regions of OR5T2, particularly the C-terminal region which shows greater sequence divergence among olfactory receptors .

• Combination approaches:

  • Sequential immunoprecipitation: Deplete samples of related receptors before OR5T2 detection

  • Two-antibody validation: Use two different OR5T2 antibodies targeting distinct epitopes

  • Functional correlation: Combine antibody detection with functional assays specific to OR5T2

• Genetic approaches as controls:

  • Use OR5T2 overexpression systems as positive controls

  • Use CRISPR/Cas9 OR5T2 knockout samples as negative controls

  • Compare detection in systems with known differential expression of OR5T2 versus related receptors

• Mass spectrometry validation: For definitive identification, consider immunoprecipitation followed by mass spectrometry to confirm antibody specificity to OR5T2 versus related receptors .

The olfactory receptor family comprises the largest gene family in the human genome, with approximately 400 functional genes, making specific detection particularly challenging and requiring rigorous validation .

How can OR5T2 antibodies be effectively used in co-immunoprecipitation studies to identify interaction partners?

Successfully using OR5T2 antibodies in co-immunoprecipitation (co-IP) studies requires careful consideration of this membrane protein's characteristics:

Methodological approach:

• Membrane protein extraction optimization:

  • Use mild detergents (e.g., digitonin, CHAPS, or DDM) to solubilize OR5T2 while preserving protein-protein interactions

  • Avoid harsh detergents like SDS which disrupt protein interactions

  • Consider crosslinking approaches to stabilize transient interactions

• Antibody selection criteria:

  • Choose antibodies validated for immunoprecipitation applications

  • Antibodies targeting extracellular loops or C-terminal regions generally perform better

  • Consider native conformation recognition capability

• Technical optimization:

  • Pre-clear lysates thoroughly to reduce non-specific binding

  • Use appropriate controls including:

    • IgG control from same species as the OR5T2 antibody

    • Immunoprecipitation from OR5T2-negative samples

    • Peptide competition controls to verify specificity

• Verification approaches:

  • Confirm successful OR5T2 pull-down by Western blot

  • Identify interaction partners using:

    • Targeted Western blot for suspected interactors

    • Mass spectrometry for unbiased partner identification

  • Validate key interactions with reverse co-IP where possible

Given that G protein-coupled receptors like OR5T2 often form transient interactions with signaling proteins, crosslinking approaches prior to lysis may significantly improve co-IP efficiency for capturing the complete interactome .

How should researchers interpret variations in OR5T2 antibody signals across different tissue types?

When interpreting variations in OR5T2 antibody signals across tissue types, researchers should consider multiple biological and technical factors:

Biological considerations:

• Expression pattern verification: OR5T2, like many olfactory receptors, shows a highly tissue-specific expression pattern, primarily in olfactory epithelium. Detection in other tissues should be carefully validated through multiple methods .

• Splice variant analysis: Check if observed molecular weight variations correspond to known OR5T2 splice variants or post-translational modifications that may be tissue-specific.

• Receptor trafficking differences: Variations in subcellular localization may reflect biological differences in receptor processing rather than technical artifacts .

Technical considerations:

• Signal normalization approach: When comparing across tissues:

  • Use appropriate loading controls specific to each tissue type

  • Consider using membrane protein-specific loading controls for more accurate normalization

  • Quantify signal relative to total protein staining methods (e.g., Ponceau S)

• Sample preparation effects: Different tissues may require different extraction methods:

  • Lipid-rich tissues may show altered OR5T2 extraction efficiency

  • Proteolytic activity varies between tissues and may affect detection

  • Fixation conditions for immunohistochemistry should be optimized for each tissue type

• Antibody validation per tissue: Confirm specificity in each tissue type using:

  • Peptide competition assays

  • Genetic models (knockout controls where available)

  • Correlation with mRNA expression data

What are the potential sources of data inconsistency when comparing results from different OR5T2 antibodies?

When comparing results obtained with different OR5T2 antibodies, researchers should consider these potential sources of inconsistency:

• Epitope differences:

  • Antibodies targeting different regions of OR5T2 may have inherently different accessibility to the epitope

  • C-terminal vs. extracellular loop antibodies may reveal different aspects of receptor biology

  • Some epitopes may be masked by protein interactions or conformational states

• Technical specifications:

  • Antibody format variations: Polyclonal vs. monoclonal antibodies have different specificity profiles

  • Host species effects: Rabbit polyclonals may have different background patterns than those from other species

  • Clonality impacts: Polyclonal antibodies recognize multiple epitopes while monoclonals target single epitopes

• Validation status discrepancies:

  • Differential validation depth across antibodies (western blot only vs. comprehensive validation)

  • Variation in negative controls used during validation

  • Different reference standards used to confirm specificity

• Protocol optimization requirements:

  Antibody Parameter	Impact on Results	Mitigation Strategy
  Affinity variations	Signal intensity differences	Optimize concentration for each antibody
  Buffer compatibility	Background differences	Customize blocking and wash conditions
  Incubation requirements	Signal-to-noise ratio differences	Adjust time and temperature parameters
  Storage stability	Batch-to-batch variations	Document lot numbers and standardize handling

How can researchers differentiate between specific OR5T2 detection and common artifacts in immunohistochemistry and immunofluorescence applications?

Distinguishing between genuine OR5T2 signal and artifacts in IHC/IF applications requires a methodical approach to validation and control experiments:

Common artifacts and their resolution:

• Membrane protein fixation artifacts:

  • Problem: Overfixation can mask OR5T2 epitopes

  • Solution: Optimize fixation time and test multiple fixatives; consider antigen retrieval methods specific for membrane proteins

  • Validation: Compare multiple fixation protocols to identify optimal signal-to-noise ratio

• Autofluorescence interference:

  • Problem: Tissues like olfactory epithelium can have high intrinsic autofluorescence

  • Solution: Use spectral unmixing or specific autofluorescence quenching reagents

  • Validation: Include unstained tissue controls to establish autofluorescence baseline

• Non-specific binding patterns:

  • Problem: Antibodies may bind to other G-protein coupled receptors with structural similarity

  • Solution: Include peptide competition controls and OR5T2-deficient samples

  • Validation: The pattern should disappear in competition assays and be absent in negative control tissues

Validation checklist for confirming authentic OR5T2 detection:

• Pattern consistency check:

  • OR5T2 should show membrane-associated localization consistent with its biology as a multi-pass membrane protein

  • Expected subcellular distribution should align with known GPCR trafficking patterns

• Multiple antibody verification:

  • Use two or more antibodies targeting different OR5T2 epitopes

  • Matching patterns strongly suggest specific detection

  • Differential patterns require further investigation

• Correlation with functional or molecular data:

  • Compare protein detection with mRNA expression data from the same tissues

  • Align with functional studies where available

  • Consider known physiological roles when interpreting localization patterns

• Technical controls to implement:

  • Secondary antibody-only controls to rule out non-specific secondary binding

  • Isotype controls to address non-specific binding of primary antibody

  • Tissue-specific positive controls with known OR5T2 expression

How are OR5T2 antibodies being utilized in receptor trafficking and internalization studies?

OR5T2 antibodies are increasingly being applied to study the dynamic processes of receptor trafficking and internalization, providing insights into olfactory receptor regulation:

Methodological approaches:

• Live-cell imaging applications:

  • Surface-specific OR5T2 antibodies (targeting extracellular domains) allow monitoring of receptor internalization in real-time

  • Pulse-chase experiments with differentially labeled antibodies can distinguish surface from internalized populations

  • Antibody feeding assays can quantify internalization rates under various stimulation conditions

• Subcellular fractionation analysis:

  • Combine with subcellular fractionation to track receptor movement between membrane and intracellular compartments

  • Quantitative western blotting of fractions can determine relative distribution changes upon stimulus

  • Co-localization with compartment markers verifies trafficking pathways

• Co-localization studies with trafficking machinery:

  • Double immunofluorescence with markers for:

    • Clathrin-coated pits (early endocytosis)

    • Early endosomes (EEA1)

    • Recycling endosomes (Rab11)

    • Lysosomes (LAMP1)

  • Provides insights into OR5T2 post-endocytic fate

While these applications show promise, researchers should be aware that antibody binding itself may potentially influence receptor trafficking, necessitating complementary approaches like epitope-tagged OR5T2 constructs to validate findings .

What are the considerations for using OR5T2 antibodies in high-throughput screening or array-based proteomics studies?

Implementing OR5T2 antibodies in high-throughput screening or proteomics arrays requires addressing several technical and analytical considerations:

Technical implementation:

• Antibody selection criteria for high-throughput applications:

  • Prioritize antibodies with:

    • High specificity (validated across multiple applications)

    • Good lot-to-lot consistency

    • Compatibility with automated systems

    • Established performance in similar high-throughput platforms

• Platform-specific optimization requirements:

  • Protein arrays: May require modified coating buffers to properly orient the hydrophobic OR5T2

  • Multiplexed assays: Need spectral compatibility assessment when using fluorescently-conjugated antibodies

  • Automated systems: Require standardized protocols with robust performance across varying conditions

• Quality control implementation:

  Parameter	Monitoring Approach	Acceptance Criteria
  Signal linearity	Titration of recombinant OR5T2	R² > 0.95 across dynamic range
  Reproducibility	Coefficient of variation	CV < 15% for technical replicates
  Specificity	Cross-reactivity panel	< 10% signal with related receptors
  Sensitivity	LOD determination	Appropriate for expected expression levels

Analytical considerations:

• Data normalization strategies:

  • Include internal reference standards on each plate/array

  • Consider global normalization methods appropriate for membrane proteins

  • Implement batch effect correction algorithms for multi-plate studies

• Result verification workflow:

  • Establish orthogonal validation for novel or unexpected findings

  • Design appropriate follow-up experiments for hits from screening

  • Consider the impact of detergents and solubilization methods on epitope accessibility

• Bioinformatic integration:

  • Correlate antibody-based detection with transcriptomic data

  • Map findings to relevant olfactory signaling pathways

  • Apply appropriate statistical methods for large-scale antibody-based datasets

These considerations help ensure reliable, reproducible results when scaling up OR5T2 antibody applications to high-throughput formats .

How do conformational changes in OR5T2 impact antibody binding, and what approaches can address conformation-dependent epitope accessibility?

As a G protein-coupled receptor, OR5T2 undergoes conformational changes during signaling, which can significantly affect antibody epitope accessibility and binding characteristics:

Impact of conformational states:

• State-dependent epitope masking:

  • Ligand binding can induce conformational changes that alter antibody epitope accessibility

  • G-protein coupling can obscure intracellular epitopes

  • Receptor dimerization may mask interaction surfaces

• Functional implications of antibody binding:

  • Some antibodies may preferentially recognize active or inactive conformations

  • Antibody binding itself may stabilize particular conformational states

  • Epitope location affects likelihood of functional interference

Methodological solutions:

• Conformation-specific antibody approaches:

  Strategy	Implementation	Application Value
  Conformational fixation	Chemical crosslinking to stabilize specific states	Capturing transient signaling states
  State-specific immunization	Generate antibodies against activated-state peptides	Measuring receptor activation
  Nanobody development	Single-domain antibodies with access to cryptic epitopes	Less disruptive to native structure

• Modified sample preparation techniques:

  • Native versus denaturing conditions in western blotting

  • Mild detergent panels to preserve conformational integrity

  • Temperature-controlled immunoprecipitation to maintain native folding

• Functional correlation analysis:

  • Combine antibody detection with functional calcium signaling assays

  • Correlate binding patterns with ligand-induced conformational changes

  • Use site-directed mutagenesis to evaluate epitope accessibility in different receptor states

Understanding these conformation-dependent factors is particularly important when using OR5T2 antibodies to study receptor activation mechanisms or for developing screening assays for olfactory receptor modulators .

How might advances in recombinant antibody technology improve OR5T2-targeted research tools?

Emerging recombinant antibody technologies offer promising opportunities to overcome current limitations in OR5T2 research:

Next-generation OR5T2 antibody development:

• Single-domain antibodies (nanobodies):

  • Smaller size allows access to cryptic epitopes in the transmembrane regions of OR5T2

  • Enhanced penetration in tissue samples improves detection in complex structures

  • Reduced interference with receptor function enables live-cell functional studies

  • Greater stability in detergent conditions facilitates membrane protein applications

• Synthetic antibody libraries:

  • Phage display selection against native OR5T2 conformation

  • Yeast display technologies for identifying high-affinity binders

  • Ribosome display approaches for generating diverse OR5T2-specific antibodies

  • These methods can yield antibodies with precisely defined epitopes and binding characteristics

• Multispecific antibody formats:

  • Bispecific antibodies targeting OR5T2 and signaling partners simultaneously

  • Antibody-based proximity labeling tools for identifying transient interactions

  • Antibody-fluorophore conjugates with improved signal-to-noise ratios

  • These tools enable more sophisticated studies of receptor complexes and signaling dynamics

Anticipated research impacts:

Enhanced research capabilities will likely emerge in several areas:

• More precise tracking of OR5T2 trafficking pathways

• Improved detection of conformational changes during receptor activation

• Better discrimination between OR5T2 and closely related olfactory receptors

• More sensitive detection of low-abundance receptor populations

What are the methodological challenges in developing quantitative assays for OR5T2 receptor density or expression levels?

Developing truly quantitative assays for OR5T2 receptor density presents several methodological challenges that require specialized approaches:

Current limitations:

• Reference standard challenges:

  • Limited availability of purified recombinant OR5T2 protein standards

  • Difficult quantitative extraction of membrane proteins like OR5T2

  • Variability in extraction efficiency across sample types

  • These factors complicate absolute quantification efforts

• Technical barriers:

  • Hydrophobic nature of OR5T2 affects consistent antibody access

  • Post-translational modifications may alter antibody recognition

  • Detergent requirements complicate assay standardization

  • Conformation-dependent epitope accessibility introduces variability

Advanced methodological solutions:

• Mass spectrometry-based approaches:

  • Targeted proteomics using multiple reaction monitoring (MRM)

  • Stable isotope-labeled internal standards for absolute quantification

  • Parallel reaction monitoring for increased sensitivity

  • These methods offer antibody-independent quantification options

• Surface quantification strategies:

  • Saturation binding assays with radiolabeled antibodies

  • Flow cytometry-based receptor quantification using calibrated standards

  • Single-molecule localization microscopy for direct counting

  • These approaches can determine actual receptor numbers rather than relative expression

• Quantitative immunoassay development:

  Method	Key Requirements	Quantification Principle
  Calibrated ELISA	Purified OR5T2 standard curve	Signal correlation to known concentrations
  Competitive binding	Labeled reference ligand	Displacement by unlabeled receptor
  Digital ELISA (Simoa)	Single-molecule detection	Counting of individual binding events

Researchers working on quantitative OR5T2 assays should consider combining orthogonal approaches to validate findings and establish reliable quantification systems for this challenging membrane protein .

What considerations should guide research into cross-reactivity between OR5T2 antibodies and other olfactory receptor family members?

Investigating cross-reactivity between OR5T2 antibodies and other olfactory receptor family members requires systematic approaches due to the high sequence similarity within this large receptor family:

Sequence-based analysis framework:

• Epitope mapping and cross-reactivity prediction:

  • Perform detailed sequence alignments between OR5T2 immunogen sequences and other olfactory receptors

  • Identify regions of high similarity that may promote cross-reactivity

  • Calculate sequence identity percentages within epitope regions

  • Prioritize testing against receptors with >70% epitope region homology

• Structural considerations affecting cross-reactivity:

  • Analyze predicted 3D structures of OR5T2 versus related receptors

  • Identify conformational epitopes that may differ despite sequence similarity

  • Consider post-translational modifications that may differentiate otherwise similar epitopes

  • Evaluate surface accessibility of potentially cross-reactive regions

Experimental validation strategy:

• Comprehensive cross-reactivity testing panel:

  • Recombinant expression of closely related olfactory receptors

  • ELISA-based screening against purified receptor proteins

  • Western blot analysis against tissue samples with differential expression profiles

  • Immunoprecipitation followed by mass spectrometry to identify all captured proteins

• Genetic approach to specificity determination:

  • Heterologous expression systems with individual olfactory receptors

  • CRISPR/Cas9 knockout of OR5T2 to confirm signal elimination

  • Overexpression systems to evaluate signal intensity correlation with expression level

  • These approaches provide definitive evidence of antibody specificity

• Application-specific cross-reactivity assessment:

  Application	Critical Cross-Reactivity Tests	Acceptance Criteria
  Western blot	Panel testing against related ORs	Single band at expected MW
  Immunohistochemistry	Staining pattern in control tissues	Pattern matches known OR5T2 distribution
  Flow cytometry	Testing against cells expressing specific ORs	Signal proportional to OR5T2 expression only

Understanding the extent and pattern of cross-reactivity is essential for correct interpretation of experimental results, especially in tissues expressing multiple olfactory receptors simultaneously .