OR1D5/OR1D4 Antibody

What are OR1D5 and OR1D4 proteins and what is their function?

OR1D5 and OR1D4 are olfactory receptor proteins belonging to the G-protein-coupled receptor (GPCR) family. They feature characteristic 7-transmembrane domain structures similar to many neurotransmitter and hormone receptors. Functionally, these receptors interact with odorant molecules in the nasal cavity to initiate neuronal responses that trigger smell perception . OR1D5 is also known as Olfactory receptor 17-31, while OR1D4 is alternatively called Olfactory receptor 17-30 . These proteins are part of the largest gene family in the human genome, with OR1D5 corresponding to UniProt ID P58170 and OR1D4 to P47884 . The nomenclature follows a systematic approach where "OR" designates an olfactory receptor, "1" indicates the family, "D" refers to the subfamily, and "4" or "5" identifies the individual member within that subfamily.

What applications are OR1D5/OR1D4 antibodies validated for in research?

OR1D5/OR1D4 antibodies have been validated for multiple research applications, with optimization parameters varying by technique:

When selecting the appropriate application, researchers should consider that performance may vary between different antibody preparations. For optimal results, validation in your specific experimental system is recommended, particularly for applications not explicitly tested by the manufacturer.

How should samples be prepared for optimal OR1D5/OR1D4 detection?

For optimal detection of OR1D5/OR1D4 using antibody-based methods, sample preparation should account for the membrane-bound nature of these GPCRs:

For Western Blot:

• Extract proteins using membrane protein extraction buffer containing mild detergents (e.g., 1% Triton X-100 or CHAPS)

• Avoid boiling samples to prevent aggregation of transmembrane proteins; instead, incubate at 37°C for 30 minutes

• Use reducing conditions with freshly prepared DTT or β-mercaptoethanol

• Run samples on 10-12% SDS-PAGE gels optimized for membrane proteins

• Transfer to PVDF membranes (preferred over nitrocellulose for hydrophobic proteins)

• Block with 5% BSA rather than milk (which can contain endogenous GPCRs)

• Apply antibody at recommended dilutions (1:500-1:2000)

For Immunofluorescence:

• Fix cells with 4% paraformaldehyde (10-15 minutes at room temperature)

• Permeabilize with 0.1-0.3% Triton X-100 (5-10 minutes)

• Block with 1-3% BSA in PBS

• Incubate with primary antibody at recommended dilutions (1:200-1:1000)

• Use appropriate fluorophore-conjugated secondary antibodies

These protocols may require optimization depending on the specific tissue or cell type being studied, particularly for tissues with known olfactory receptor expression.

How can OR1D5/OR1D4 antibodies be validated for specificity in olfactory research?

Validating OR1D5/OR1D4 antibody specificity requires a multi-faceted approach addressing the particular challenges of olfactory receptor detection:

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess OR1D4/OR1D5 blocking peptide

  • Compare results with and without peptide competition

  • Specific signals should be significantly reduced or eliminated with peptide competition

• Heterologous Expression System Validation:

  • Express recombinant OR1D5 (available as recombinant proteins) or OR1D4 individually in cell lines that don't endogenously express olfactory receptors (e.g., HEK293)

  • Test antibody against both transfected and non-transfected cells

  • Use epitope-tagged constructs (e.g., FLAG, HA) for co-localization studies

• Genetic Validation:

  • Implement CRISPR/Cas9 knockout or siRNA knockdown of OR1D5/OR1D4 in relevant cell models

  • Compare antibody signal in wild-type versus knockout/knockdown samples

  • Quantify reduction in signal corresponding to decreased expression

• Cross-Reactivity Assessment:

  • Test against closely related olfactory receptors in the same subfamily

  • Particularly important when distinguishing between OR1D5 and OR1D4 due to their sequence similarity

  • Use recombinant proteins representing different olfactory receptor subtypes

This methodical validation strategy ensures that experimental observations accurately reflect OR1D5/OR1D4 biology rather than technical artifacts or cross-reactivity with other olfactory receptors.

What are the optimal conditions for using OR1D5/OR1D4 antibodies in co-immunoprecipitation experiments?

Optimizing co-immunoprecipitation (Co-IP) experiments with OR1D5/OR1D4 antibodies requires special consideration due to the hydrophobic nature of these transmembrane proteins:

• Lysis Buffer Optimization:

  • Use mild detergents to solubilize membrane proteins while preserving protein-protein interactions

  • Recommended formula: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40 or 0.5-1% digitonin, supplemented with protease inhibitors

  • Avoid harsh detergents like SDS that disrupt protein interactions

• Antibody Selection and Coupling:

  • Choose affinity-purified antibodies (as indicated for most OR1D5/OR1D4 antibodies)

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Consider covalently coupling the antibody to beads using dimethyl pimelimidate (DMP) to prevent antibody co-elution

• Incubation Parameters:

  • Perform binding reactions at 4°C overnight with gentle rotation

  • Maintain protein concentration at 1-2 mg/ml for optimal signal-to-noise ratio

  • Include appropriate controls: non-immune IgG, lysates from cells not expressing OR1D5/OR1D4

• Washing Conditions:

  • Use milder washing conditions than standard Co-IP protocols

  • Gradually increase wash stringency to find optimal balance between background reduction and maintenance of specific interactions

  • Example washing buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% detergent

This methodological approach accounts for the challenges of working with GPCRs in co-immunoprecipitation experiments while maximizing the chances of detecting physiologically relevant interactions.

How can OR1D5/OR1D4 antibodies be used to study receptor trafficking?

Investigating olfactory receptor trafficking with OR1D5/OR1D4 antibodies requires methodologies that capture the dynamic nature of receptor movement within cells:

• Pulse-Chase Immunofluorescence:

  • Label cell surface receptors using non-permeabilized immunostaining with OR1D5/OR1D4 antibodies

  • Allow internalization for varying time periods

  • Permeabilize and detect total receptor pool with different fluorophore

  • Quantify internalization rates and trafficking patterns

• Subcellular Fractionation Analysis:

  • Separate cellular compartments (plasma membrane, endoplasmic reticulum, Golgi, endosomes)

  • Perform Western blot with OR1D5/OR1D4 antibodies (1:500-1:2000)

  • Quantify relative distribution across compartments under basal and stimulated conditions

  • Include compartment-specific markers as controls

• Co-localization with Trafficking Machinery:

  • Perform dual immunofluorescence with OR1D5/OR1D4 antibodies (1:200-1:1000) and markers for:

    • Endoplasmic reticulum (e.g., calnexin, KDEL)

    • Golgi apparatus (e.g., GM130, TGN46)

    • Endosomes (e.g., Rab5, Rab7, Rab11)

    • Recycling pathways (e.g., transferrin receptor)

  • Analyze co-localization using quantitative image analysis

• Live Cell Trafficking Studies:

  • Validate antibody-based observations using GFP-tagged OR1D5/OR1D4 constructs

  • Use recombinant antibody fragments (Fab, scFv) conjugated to fluorophores for live tracking

  • Correlate live observations with fixed-cell antibody staining patterns

This methodological framework enables researchers to decipher the complex trafficking pathways of OR1D5/OR1D4 from biosynthesis to internalization and recycling, providing insights into the regulation of olfactory sensing.

What techniques enhance OR1D5/OR1D4 detection in tissues with low expression?

Detecting OR1D5/OR1D4 in tissues with low expression requires methodological refinements to enhance sensitivity while maintaining specificity:

• Signal Amplification Strategies:

  • Implement tyramide signal amplification (TSA) for immunohistochemistry and immunofluorescence

  • Use high-sensitivity chemiluminescent substrates for Western blot (e.g., SuperSignal West Femto)

  • Consider biotin-streptavidin amplification systems with biotinylated secondary antibodies

• Sample Enrichment Techniques:

  • For protein extracts, use immunoprecipitation with OR1D5/OR1D4 antibodies before Western blot

  • Implement laser capture microdissection to isolate regions with known expression

  • Use membrane protein enrichment protocols to concentrate transmembrane receptors

• Antibody Selection and Optimization:

  • Choose antibodies with demonstrated highest sensitivity (1:20000 dilution capability in ELISA suggests high affinity)

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

  • Optimize blocking conditions to reduce background while preserving specific signal

• Technical Controls:

  • Include positive controls with known OR1D5/OR1D4 expression (e.g., recombinant proteins)

  • Use blocking peptides to confirm signal specificity

  • Implement parallel detection methods (e.g., RNAscope for mRNA detection alongside protein detection)

This methodological framework enables detection of OR1D5/OR1D4 even in challenging samples with expression levels at the lower limits of conventional detection methods.

How should antibody dilution be optimized across samples with variable expression?

Optimizing antibody dilution for OR1D5/OR1D4 detection across samples with variable expression requires a systematic approach:

• Antibody Titration Strategy:

  • Perform serial dilution series (e.g., 1:100, 1:200, 1:500, 1:1000, 1:2000)

  • Test across multiple sample types (high and low expressing)

  • Plot signal-to-noise ratio against antibody concentration

  • Identify dilution with maximal specific signal and minimal background

• Sample-Dependent Adjustment:

  • For Western blot:

    • High expression samples: Use higher dilutions (1:1000-1:2000)

    • Low expression samples: Use lower dilutions (1:500)

  • For immunofluorescence:

    • Adjust from recommended range (1:200-1:1000) based on expression

    • Consider longer incubation times for lower antibody concentrations

• Quantitative Calibration:

  • Create standard curves using recombinant OR1D5 at known concentrations

  • Establish detection thresholds for different antibody dilutions

  • Implement internal loading controls for normalization

  • Use digital image analysis to quantify signal intensities

• Technical Considerations:

  • Prepare fresh antibody dilutions for each experiment

  • Store antibodies according to manufacturer recommendations (commonly at -20°C with 50% glycerol, 0.5% BSA, and 0.02% sodium azide)

  • Avoid repeated freeze-thaw cycles

  • For critical applications, aliquot antibody stock to maintain consistency

This methodological approach ensures optimal detection of OR1D5/OR1D4 across different experimental contexts while maintaining specificity and reproducibility.

How can non-specific binding be reduced when using OR1D5/OR1D4 antibodies?

Non-specific binding presents a significant challenge when working with OR1D5/OR1D4 antibodies. A methodical approach to reducing background while preserving specific signal includes:

• Validation with Blocking Peptides:

  • Implement OR1D5/OR1D4 blocking peptides (available commercially) in parallel experiments

  • Pre-incubate antibody with excess peptide (5-10x molar ratio)

  • Bands or signals that disappear in peptide-blocked samples represent specific binding

  • Persistent signals likely represent non-specific binding

• Blocking Optimization:

  • Test different blocking agents (5% BSA often superior to milk for GPCR detection)

  • Extend blocking time (2 hours at room temperature or overnight at 4°C)

  • Add 0.1-0.3% Triton X-100 to blocking buffer to reduce hydrophobic interactions

  • Include 5% normal serum from the secondary antibody host species

• Washing Protocol Refinement:

  • Increase number of wash steps (5-6 washes of 5-10 minutes each)

  • Use higher detergent concentrations in wash buffers (0.1-0.3% Tween-20)

  • Implement more stringent wash buffers for problematic samples (add 500mM NaCl)

  • Extend final wash duration before detection

• Antibody Dilution Optimization:

  • Titrate antibodies to determine optimal concentration

  • Prepare antibody dilutions in fresh buffer containing 1-3% BSA

  • Pre-adsorb antibodies against tissues or cells lacking target protein

  • Centrifuge diluted antibody before use to remove aggregates

These methodological refinements allow researchers to distinguish true OR1D5/OR1D4 signal from non-specific background, increasing confidence in experimental results.

What approaches resolve discrepancies between Western blot and immunohistochemistry results?

Discrepancies between Western blot and immunohistochemistry results with OR1D5/OR1D4 antibodies require systematic troubleshooting to reconcile these differences:

• Epitope Accessibility Analysis:

  • In Western blot, proteins are denatured, exposing all epitopes

  • In IHC/IF, proteins retain native conformation and membrane integration

  • Solution: Try multiple fixation and permeabilization protocols for IHC/IF

  • Consider native vs. denaturing conditions for Western blot

• Cross-Validation Strategies:

  • Implement antibodies targeting different epitopes of OR1D5/OR1D4

  • Compare results from multiple antibodies to identify consistent patterns

  • Correlate with mRNA expression (RT-PCR, in situ hybridization)

  • Use transfected cells expressing recombinant OR1D5 as positive controls

• Method-Specific Optimization:

  • For Western blot:

    • Adjust protein denaturation conditions (temperature, reducing agents)

    • Try native PAGE for membrane proteins

    • Optimize transfer conditions for hydrophobic proteins

  • For IHC/IF:

    • Implement antigen retrieval methods (heat-induced, enzyme-based)

    • Test detergent permeabilization parameters

    • Adjust antibody incubation times and temperatures

• Context-Dependent Expression:

  • Document sample source and preparation differences

  • Consider that protein modifications may differ between samples

  • Evaluate potential tissue-specific interacting proteins that might mask epitopes

  • Assess protein degradation in different sample types

This methodological framework helps researchers understand the basis for discrepancies between different detection methods and develop a coherent interpretation of OR1D5/OR1D4 expression patterns.

What alternatives exist when OR1D5/OR1D4 antibodies fail to detect target proteins?

When conventional OR1D5/OR1D4 antibody approaches yield unsatisfactory results, researchers can implement alternative detection strategies:

• Alternative Detection Technologies:

  • RNAscope in situ hybridization for OR1D5/OR1D4 mRNA with single-molecule sensitivity

  • Mass spectrometry-based proteomics targeting OR1D5/OR1D4-specific peptides

  • Proximity ligation assay with combinations of antibodies targeting different epitopes

  • CRISPR-based endogenous tagging with epitope tags (FLAG, HA, V5)

• Recombinant Expression Systems:

  • Utilize available recombinant OR1D5 proteins for comparative studies

  • Generate tissue-specific viral expression systems

  • Develop reporter gene constructs under control of OR1D5/OR1D4 promoters

  • Create cell lines with controlled expression for methodological optimization

• Specialized Antibody Approaches:

  • Test multiple commercially available antibodies targeting different epitopes

  • Consider custom antibody development with tissue-specific optimization

  • Implement antibody fragments (Fab, F(ab')2) to improve tissue penetration

  • Use nanodisc-embedded proteins as immunogens for improved conformational epitopes

These alternative methodological approaches provide researchers with options when conventional antibody-based detection proves challenging, ensuring continued investigation of OR1D5/OR1D4 biology across diverse experimental contexts.

How can researchers distinguish between OR1D5 and OR1D4 in experimental systems?

Despite their sequence similarity, differentiating between OR1D5 and OR1D4 can be achieved through these methodological approaches:

• Selective Antibody Screening:

  • Test multiple antibodies to identify those with preferential binding to either OR1D5 or OR1D4

  • Validate selectivity using recombinant proteins expressing each receptor individually

  • Implement epitope mapping to identify unique recognition sites

  • Use competitive binding assays to quantify relative affinities

• Genetic Discrimination Techniques:

  • Design isoform-specific RT-PCR primers targeting divergent nucleotide sequences

  • Implement CRISPR/Cas9 knockout of individual receptors

  • Use RNA interference with highly specific siRNA sequences

  • Apply differential gene expression analysis (RNA-seq) with isoform-level resolution

• Functional Discrimination:

  • Identify ligands with differential activation profiles between OR1D5 and OR1D4

  • Measure receptor-specific downstream signaling pathways

  • Implement calcium imaging with receptor-specific expression systems

  • Analyze differential trafficking patterns between the two receptors

• Computational Analysis:

  • Apply bioinformatic tools to identify unique structural features

  • Develop machine learning algorithms for automated detection in imaging data

  • Implement molecular dynamics simulations to predict functional differences

  • Use quantitative image analysis for subtle differences in localization patterns

These methodological approaches enable researchers to overcome the challenge of differentiating between these highly homologous receptors in their experimental systems.