OR1D4/OR1D5 Antibody

What are OR1D4 and OR1D5 receptors?

OR1D4 and OR1D5 are olfactory receptors belonging to the G-protein-coupled receptor (GPCR) family. These receptors interact with odorant molecules in the nose to initiate neuronal responses that trigger smell perception. They share a 7-transmembrane domain structure common to many neurotransmitter and hormone receptors and are responsible for the recognition and G protein-mediated transduction of odorant signals . The genes encoding these receptors are part of the largest gene family in the human genome. OR1D4 is also known as Olfactory receptor 17-30 (OR17-30), while OR1D5 is also known as Olfactory receptor 17-31 (OR17-31) .

What is the molecular weight of OR1D4/OR1D5 proteins?

The molecular weight of OR1D4/OR1D5 proteins is approximately 35,227 Da . This information is critical when analyzing Western blot results to confirm the specificity of antibody binding. When performing SDS-PAGE and subsequent Western blotting, researchers should expect to observe bands corresponding to this molecular weight. Variations from this expected weight might indicate post-translational modifications, protein degradation, or non-specific binding that would require further optimization of experimental protocols .

What applications are OR1D4/OR1D5 antibodies suitable for?

OR1D4/OR1D5 antibodies have been validated for multiple research applications including:

These antibodies have been successfully used to detect OR1D4/OR1D5 expression in various cell lines and can be employed in research focusing on olfactory receptor distribution and function .

Which cell lines have been validated for OR1D4/OR1D5 antibody use?

The OR1D4/OR1D5 antibodies have been successfully tested on multiple cell lines for Western blot analysis, including:

• HeLa (human cervical cancer cells)

• HUVEC (human umbilical vein endothelial cells)

• Jurkat (human T lymphocyte cells)

• HepG2 (human liver cancer cells)

• MCF-7 (human breast cancer cells)

• K562 (human myelogenous leukemia cells)

This validation across diverse cell types suggests the antibody's reliability for detecting these receptors in various research contexts, including both cancer and non-cancer cell lines .

How can I distinguish between OR1D4 and OR1D5 in my experiments?

Distinguishing between OR1D4 and OR1D5 presents a significant challenge due to their high sequence homology. Most commercially available antibodies recognize both receptors due to shared epitopes . For experiments requiring specific detection of either OR1D4 or OR1D5:

• Consider using RNA-based methods (qPCR with specific primers) alongside protein detection.

• Perform epitope mapping to identify regions with sequence differences.

• Use blocking peptides specific to each receptor to confirm antibody specificity.

• Consider knockout/knockdown controls for each receptor separately.

When absolute distinction is critical, custom antibodies raised against unique peptide sequences from each receptor may be necessary, though this approach requires extensive validation .

What controls should be included when validating OR1D4/OR1D5 antibody specificity?

A robust validation strategy for OR1D4/OR1D5 antibodies should include:

• Positive control: Cell lines known to express OR1D4/OR1D5 (e.g., MCF-7, HeLa)

• Negative control: Cell lines with minimal expression or OR1D4/OR1D5-knockout models

• Peptide competition: Pre-incubating the antibody with the immunizing peptide should eliminate specific binding

• Secondary antibody-only control: To detect non-specific binding from the secondary antibody

• Isotype control: Using matched IgG from the same species to identify non-specific binding

Evidence from the literature shows successful peptide competition assays with OR1D4/OR1D5 antibodies in Western blot and immunofluorescence applications, where signal disappears when the antibody is pre-incubated with the synthesized peptide .

How can I optimize Western blot protocols for OR1D4/OR1D5 detection?

Optimizing Western blot protocols for OR1D4/OR1D5 detection requires careful consideration of several parameters:

• Sample preparation:

  • Use fresh samples or properly stored frozen lysates

  • Include protease inhibitors to prevent degradation

  • Consider membrane-enriched fractions as these are membrane proteins

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal separation around 35 kDa

  • Load sufficient protein (20-50 μg total protein per lane)

• Transfer and blocking:

  • Use PVDF membranes for better protein retention

  • Block with 5% BSA rather than milk (GPCRs can interact non-specifically with milk proteins)

• Antibody incubation:

  • Start with 1:1000 dilution in 5% BSA/TBST

  • Incubate primary antibody overnight at 4°C

  • Use validated cell lines (HeLa, MCF-7) as positive controls

• Detection:

  • Use enhanced chemiluminescence with appropriate exposure times

  • Consider gradual dilutions if signal is too strong or weak

Western blot analysis of lysates from multiple cell lines shows clear bands at the expected molecular weight when these optimization steps are followed .

What are the methodological considerations for immunofluorescence with OR1D4/OR1D5 antibodies?

For successful immunofluorescence studies with OR1D4/OR1D5 antibodies:

• Fixation and permeabilization:

  • 4% paraformaldehyde (10-15 minutes) preserves membrane protein structure

  • Mild permeabilization with 0.1-0.2% Triton X-100 is recommended

  • Overfixation can mask epitopes of membrane proteins

• Antibody dilution and incubation:

  • Start with 1:500 dilution based on validated protocols

  • Incubate primary antibody overnight at 4°C for optimal binding

  • Include parallel samples with blocking peptide as specificity controls

• Visualization and colocalization:

  • Use confocal microscopy for precise subcellular localization

  • Consider counterstaining with membrane markers to confirm localization

  • Z-stack imaging helps distinguish true membrane localization from cytoplasmic signals

• Controls and validation:

  • Include peptide competition controls

  • Use cell lines with known expression patterns (e.g., MCF-7)

Published images show that OR1D4/OR1D5 antibodies typically reveal a membrane and cytoplasmic distribution pattern in positive cell lines, with signal effectively blocked by competing peptides .

How do expression patterns of OR1D4/OR1D5 differ across tissue types?

OR1D4/OR1D5 expression was initially characterized in olfactory epithelium, but research has revealed expression in diverse tissues:

When investigating expression in non-olfactory tissues, it's essential to use multiple detection methods (protein and mRNA) due to potentially low expression levels. The validation of OR1D4/OR1D5 antibodies in diverse cell lines including HeLa, HUVEC, Jurkat, HepG2, and MCF-7 suggests expression across multiple tissue types, though at varying levels .

What approaches can address non-specific binding when using OR1D4/OR1D5 antibodies?

Non-specific binding is a common challenge with OR1D4/OR1D5 antibodies. To mitigate this issue:

• Optimize blocking conditions:

  • Use 5% BSA instead of milk

  • Consider adding 0.1-0.5% Tween-20 to reduce hydrophobic interactions

  • Extended blocking (2+ hours) may reduce background

• Antibody optimization:

  • Titrate antibody concentrations starting at higher dilutions (1:2000)

  • Reduce incubation temperature (4°C) and extend time (overnight)

  • Pre-absorb antibody with cell/tissue lysates from negative controls

• Stringent washing:

  • Increase washing duration and volume

  • Use PBS-T with higher detergent concentration (0.1-0.2% Tween-20)

  • Include additional washing steps after primary and secondary antibody incubations

• Signal validation:

  • Always run peptide competition controls

  • Compare observed bands/signals with expected molecular weight

  • Use gradient gels to better resolve proteins of similar sizes

Western blot analysis shows that proper optimization can yield clean bands at the expected molecular weight (35 kDa), with minimal background when appropriate controls and blocking strategies are employed .

What are the best strategies for co-immunoprecipitation studies with OR1D4/OR1D5?

Co-immunoprecipitation (co-IP) with OR1D4/OR1D5 requires special considerations due to their membrane protein nature:

• Lysis buffer optimization:

  • Use non-denaturing detergents (0.5-1% NP-40, Digitonin, or CHAPS)

  • Include physiological salt concentrations (150 mM NaCl)

  • Add protease inhibitors and phosphatase inhibitors if studying phosphorylation events

• Pre-clearing strategy:

  • Pre-clear lysates with control IgG and Protein A/G beads

  • Use matched species IgG controls to identify non-specific binding

• Antibody binding:

  • Use 2-5 μg antibody per 500 μg protein lysate

  • Incubate overnight at 4°C with gentle rotation

  • Consider crosslinking antibody to beads to avoid antibody contamination in eluates

• Washing conditions:

  • Use stringent washes to minimize non-specific interactions

  • Include detergent in all wash buffers

  • Consider salt gradient washes for removing weak interactions

• Elution and analysis:

  • Elute with gentle conditions to maintain interacting protein structure

  • Analyze by Western blot with antibodies against suspected interaction partners

This methodology is particularly relevant for studies investigating G-protein coupling and other signaling partners of OR1D4/OR1D5.

How can I implement OR1D4/OR1D5 antibodies in high-throughput screening assays?

Adapting OR1D4/OR1D5 antibodies for high-throughput screening requires careful optimization:

• ELISA-based approaches:

  • Use recommended 1:20000 antibody dilution for optimal sensitivity

  • Develop sandwich ELISA protocols with capture and detection antibodies

  • Implement automated washing and detection systems

• Cell-based screening:

  • Adapt immunofluorescence protocols to 96/384-well formats

  • Optimize cell density, fixation, and antibody concentration

  • Use automated microscopy with standardized exposure settings

• Protein array applications:

  • Validate antibody specificity on protein arrays before screening

  • Optimize blocking to minimize background on array surfaces

  • Develop quantitative readout systems

• Quality control measures:

  • Include positive controls (known expressing cells) on each plate

  • Monitor batch-to-batch antibody variation

  • Implement statistically robust normalization methods

These approaches enable screening for compounds that modulate OR1D4/OR1D5 expression or function, or for identifying tissue samples with aberrant receptor expression .

What considerations should be made when using OR1D4/OR1D5 antibodies for immunohistochemistry?

Although not explicitly mentioned in the search results as a validated application, researchers interested in immunohistochemistry (IHC) with OR1D4/OR1D5 antibodies should consider:

• Tissue preparation:

  • Test both frozen and FFPE tissues as epitope accessibility may differ

  • Optimize antigen retrieval (citrate buffer pH 6.0 is often effective for GPCRs)

  • Consider light fixation to preserve membrane protein epitopes

• Protocol adaptation:

  • Start with dilutions used for immunofluorescence (1:200-1:1000)

  • Test multiple detection systems (HRP/DAB vs. fluorescent)

  • Develop tissue-specific blocking protocols

• Validation approaches:

  • Use olfactory epithelium as positive control tissue

  • Implement peptide competition controls

  • Compare with in situ hybridization for mRNA detection

• Signal interpretation:

  • Expect membrane localization with possible cytoplasmic components

  • Carefully distinguish specific staining from tissue autofluorescence

  • Quantify expression using digital image analysis with appropriate controls

These methodological considerations are extrapolated from the validated applications (WB, IF, ELISA) and general principles for GPCR detection in tissues .

How can I resolve inconsistent results when using OR1D4/OR1D5 antibodies?

Inconsistent results with OR1D4/OR1D5 antibodies may stem from several factors:

• Sample preparation variability:

  • Standardize cell culture conditions and passage numbers

  • Use consistent lysis protocols with fresh buffers

  • Aliquot antibodies to avoid freeze-thaw cycles

• Technical optimization:

  • Create detailed protocols with standardized incubation times and temperatures

  • Prepare master mixes for antibody dilutions

  • Calibrate equipment (gel systems, imagers) regularly

• Systematic approach to troubleshooting:

  • Change one variable at a time (blocking agent, antibody dilution, incubation time)

  • Document all changes and outcomes methodically

  • Run positive control samples in parallel with experimental samples

• Antibody validation:

  • Test multiple antibody lots if possible

  • Re-validate each new lot against known positive controls

  • Consider alternative antibodies targeting different epitopes

Western blot analysis of OR1D4/OR1D5 across multiple cell lines (HeLa, HUVEC, Jurkat, HepG2, MCF-7, K562) shows that consistent results can be achieved when these factors are properly controlled .

What are the best storage and handling practices for OR1D4/OR1D5 antibodies?

To maintain antibody performance and consistency:

• Storage conditions:

  • Store at -20°C for long-term preservation

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

  • Store working dilutions at 4°C for no more than 2 weeks

• Buffer considerations:

  • Commercial OR1D4/OR1D5 antibodies are typically supplied in PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide

  • Maintain these components in working dilutions when possible

  • Avoid bacterial contamination by using sterile technique

• Handling precautions:

  • Allow antibodies to equilibrate to room temperature before opening

  • Centrifuge briefly before opening to collect liquid

  • Avoid contamination of stock solutions

• Quality monitoring:

  • Record lot numbers and performance characteristics

  • Regularly test against positive controls

  • Document any changes in performance over time

Proper storage and handling ensure consistent antibody performance across experiments, particularly important for longitudinal studies .

How are OR1D4/OR1D5 antibodies used in olfactory neuroscience research?

In olfactory neuroscience, OR1D4/OR1D5 antibodies serve several important functions:

• Receptor localization studies:

  • Mapping receptor distribution within olfactory epithelium

  • Examining subcellular localization in olfactory neurons

  • Investigating developmental expression patterns

• Signal transduction research:

  • Identifying coupling with G-proteins and downstream effectors

  • Studying receptor internalization upon odorant binding

  • Examining adaptation and desensitization mechanisms

• Comparative neurobiology:

  • Comparing OR1D4/OR1D5 expression across species

  • Investigating evolutionary conservation of olfactory pathways

  • Studying receptor specialization in different ecological niches

• Clinical applications:

  • Examining receptor alterations in olfactory disorders

  • Investigating changes in receptor expression with aging

  • Exploring links between olfactory receptor expression and neurodegenerative diseases

These applications utilize the validated immunofluorescence protocols to visualize receptor distribution and Western blotting to quantify expression levels in different experimental conditions .

What methodologies can investigate OR1D4/OR1D5 involvement in non-canonical signaling pathways?

Recent research suggests olfactory receptors may function outside traditional olfactory contexts. To investigate non-canonical roles:

• Functional studies:

  • siRNA knockdown followed by signaling assays

  • Overexpression systems with reporter gene assays

  • CRISPR-Cas9 knockout models to identify phenotypic changes

• Signaling pathway analysis:

  • Phosphorylation studies of downstream signaling components

  • Calcium imaging to detect receptor activation

  • cAMP assays to measure G-protein coupling efficiency

• Protein interaction studies:

  • Co-immunoprecipitation to identify novel binding partners

  • Proximity ligation assays to confirm interactions in situ

  • Mass spectrometry of immunoprecipitated complexes

• Physiological relevance assessment:

  • Tissue-specific expression analysis using validated antibodies

  • Correlation of expression levels with physiological functions

  • Ligand screening to identify non-odorant activators

These approaches leverage the validated applications of OR1D4/OR1D5 antibodies while extending their use to novel research questions about non-olfactory functions .