OR5D16 Antibody

What is OR5D16 and why is it studied in research?

OR5D16 is a member of the olfactory receptor family 5, subfamily D, member 16. Olfactory receptors are primarily known for their role in smell detection, but research has revealed their expression in non-olfactory tissues, suggesting broader physiological functions. Studying OR5D16 can provide insights into both normal physiology and potential disease associations. Research involving OR5D16 antibodies allows for the detection, localization, and characterization of this receptor in various tissues and experimental models .

What types of OR5D16 antibodies are available for research?

Several types of OR5D16 antibodies are available for research purposes:

• Polyclonal antibodies: These are produced in rabbits and recognize multiple epitopes of the OR5D16 protein. Examples include affinity-isolated antibodies from Prestige Antibodies® powered by Atlas Antibodies .

• Region-specific antibodies: Some antibodies target specific regions of the OR5D16 protein, such as the internal region, allowing for more targeted analyses .

Most commercially available OR5D16 antibodies are unconjugated, though they can be used with secondary detection systems for various applications .

What applications are OR5D16 antibodies validated for?

OR5D16 antibodies have been validated for several research applications:

• Immunohistochemistry (IHC): Recommended dilutions typically range from 1:50 to 1:200

• Western Blotting (WB): Generally used at 1:500-1:1000 dilutions

• Immunofluorescence (IF) and Immunocytochemistry (ICC): Effective at 1:100-1:500 dilutions

• ELISA: Can be used at higher dilutions (1:20000-1:40000)

The Prestige Antibodies® line has undergone extensive validation including testing against tissue arrays of 44 normal human tissues and 20 common cancer types, as well as protein arrays of 364 human recombinant protein fragments .

How should OR5D16 antibodies be stored and handled?

For optimal performance and longevity of OR5D16 antibodies:

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

• Most are supplied in a buffered aqueous glycerol solution, which helps maintain stability

• Some formulations contain sodium azide as a preservative, which requires careful handling as it is considered hazardous

• When shipping is required, these antibodies should be transported on wet ice to maintain their activity

• Avoid repeated freeze-thaw cycles, which can degrade antibody quality and reduce performance in experimental applications

What factors should be considered when optimizing immunohistochemistry protocols with OR5D16 antibodies?

When optimizing IHC protocols with OR5D16 antibodies, researchers should consider:

• Fixation method: Different fixatives (formaldehyde, paraformaldehyde, alcohols) can affect epitope accessibility. The Prestige Antibodies® have been validated on formalin-fixed, paraffin-embedded tissues .

• Antigen retrieval: Heat-induced epitope retrieval (HIER) or enzymatic methods may be necessary to expose the epitope recognized by the OR5D16 antibody. The optimal pH and buffer composition should be determined empirically.

• Blocking strategy: Sufficient blocking is crucial to reduce non-specific binding, especially since OR5D16 antibodies are polyclonal .

• Antibody concentration: Although the recommended range is 1:50-1:200 for IHC, optimization is necessary for each tissue type and experimental condition .

• Detection system: Consider whether chromogenic or fluorescent detection is more appropriate for your research question, and select compatible secondary reagents.

• Controls: Include both positive controls (tissues known to express OR5D16) and negative controls (omission of primary antibody) to validate staining specificity.

How can specificity of OR5D16 antibodies be verified in experimental systems?

Verifying antibody specificity is crucial for reliable research results. For OR5D16 antibodies, consider these approaches:

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (DVKDAIRKIINTKYFHIKHRHWYPF for some OR5D16 antibodies) before application to samples. Specific staining should be blocked by this competition .

• Knockout/knockdown validation: Compare staining in samples with normal OR5D16 expression versus those where expression has been eliminated or reduced through genetic approaches (CRISPR/Cas9, siRNA).

• Orthogonal detection methods: Correlate protein detection by the antibody with mRNA expression data from qPCR or RNA-seq.

• Cross-reactivity assessment: Test the antibody against recombinant proteins with similar sequences. Prestige Antibodies® have been tested against protein arrays of 364 human recombinant protein fragments to ensure minimal cross-reactivity .

• Multiple antibodies approach: Use antibodies targeting different epitopes of OR5D16 and compare staining patterns; concordant results increase confidence in specificity.

What are the considerations for quantifying OR5D16 expression in Western blot analyses?

For accurate quantification of OR5D16 in Western blot analyses:

• Sample preparation:

  • Select appropriate lysis buffers that effectively solubilize membrane proteins like OR5D16

  • Include protease inhibitors to prevent degradation

  • Determine optimal protein loading amounts (typically 20-50 μg of total protein)

• Electrophoresis conditions:

  • OR5D16 is a transmembrane protein; use SDS-PAGE conditions suitable for membrane proteins

  • Consider gradient gels for better resolution

• Transfer conditions:

  • Optimize transfer time and voltage for efficient transfer of OR5D16 to membranes

  • Consider using PVDF membranes which may provide better retention of hydrophobic proteins

• Detection optimization:

  • Use the recommended dilution range (1:500-1:1000) as a starting point, then optimize

  • Select appropriate secondary antibodies with minimal background

  • Consider enhanced chemiluminescence (ECL) versus fluorescent detection based on sensitivity requirements

• Quantification controls:

  • Include loading controls appropriate for your sample type

  • Use recombinant OR5D16 protein or peptide as positive controls where available

  • Consider normalization to total protein rather than single housekeeping proteins

How can OR5D16 subcellular localization best be determined using immunofluorescence techniques?

For precise subcellular localization of OR5D16:

• Cell preparation:

  • Optimize fixation methods (paraformaldehyde is often suitable for membrane proteins)

  • Consider mild permeabilization methods to maintain membrane integrity while allowing antibody access

• Co-localization studies:

  • Use established markers for cellular compartments (plasma membrane, endoplasmic reticulum, Golgi apparatus)

  • Select fluorophores with minimal spectral overlap to reduce bleed-through

  • Include nuclear counterstains (DAPI/Hoechst) for cellular orientation

• Advanced imaging:

  • Consider confocal microscopy for improved resolution of subcellular structures

  • Super-resolution techniques (STED, STORM, PALM) may provide nanoscale localization information

  • Z-stack imaging can help distinguish membrane versus cytoplasmic localization

• Antibody optimization:

  • Use the recommended dilution range (1:100-1:500) for IF/ICC as a starting point

  • Optimize blocking conditions to reduce background fluorescence

  • Consider signal amplification methods for low-abundance targets

• Controls:

  • Include cells with known expression patterns

  • Use peptide competition controls to confirm specificity

  • Include secondary-only controls to assess background

What methodological approaches can address potential discrepancies between OR5D16 antibody results and mRNA expression data?

When facing discrepancies between protein detection with OR5D16 antibodies and mRNA expression:

• Technical considerations:

  • Verify antibody specificity using methods described in FAQ 2.2

  • Assess mRNA data quality and primer/probe specificity

  • Consider RNA and protein degradation rates in sample preparation

• Biological explanations:

  • Post-transcriptional regulation may affect OR5D16 mRNA translation efficiency

  • Post-translational modifications might alter antibody epitope recognition

  • Protein turnover rates may differ from mRNA degradation rates

• Methodological approaches:

  • Employ multiple detection methods (IHC, IF, WB) with the same antibody

  • Use different antibodies targeting distinct epitopes of OR5D16

  • Perform time-course experiments to detect temporal differences in mRNA versus protein expression

  • Consider polysome profiling to assess translation efficiency of OR5D16 mRNA

• Validation strategies:

  • Use recombinant expression systems to confirm antibody detection capacity

  • Consider absolute quantification methods for both mRNA and protein

  • Evaluate potential isoforms that might be detected differentially

How can OR5D16 antibodies be integrated into multi-omics research approaches?

Integrating OR5D16 antibodies into multi-omics research:

• Proteogenomic integration:

  • Correlate protein expression detected by OR5D16 antibodies with genomic variations in the OR5D16 gene

  • Investigate how genetic polymorphisms might affect protein expression and antibody detection

• Transcriptome-proteome correlation:

  • Compare OR5D16 mRNA expression patterns with protein localization and abundance

  • Identify potential regulatory mechanisms affecting translation efficiency

• Interactome studies:

  • Use OR5D16 antibodies for co-immunoprecipitation followed by mass spectrometry

  • Identify protein-protein interaction networks involving OR5D16

• Functional genomics:

  • Apply OR5D16 antibodies to validate CRISPR screens or other functional genomics approaches

  • Confirm knockdown/knockout efficiency at the protein level

• Spatial omics integration:

  • Combine single-cell transcriptomics with spatial proteomics using OR5D16 antibodies

  • Map tissue microenvironments where OR5D16 plays functional roles

What are best practices for using OR5D16 antibodies in tissue microarray (TMA) analysis?

When employing OR5D16 antibodies in TMA studies:

• Antibody validation for TMA application:

  • Validate on whole tissue sections before applying to TMAs

  • Determine optimal staining conditions using the recommended dilution range (1:50-1:200)

  • Include positive and negative control tissues on each TMA

• TMA design considerations:

  • Include sufficient technical replicates (multiple cores per sample)

  • Consider tissue heterogeneity when selecting core locations

  • Include orientation markers and control tissues

• Staining protocol standardization:

  • Use automated staining platforms where possible to ensure consistency

  • Implement stringent quality control measures

  • Process all TMAs in the same batch when possible

• Image analysis approaches:

  • Develop consistent scoring methods (manual or automated)

  • Consider both staining intensity and distribution patterns

  • Validate scoring consistency between observers or algorithms

• Data integration:

  • Correlate OR5D16 expression with clinical parameters and outcomes

  • Integrate with other molecular markers for comprehensive analysis

  • Consider computational methods for pattern recognition in large datasets

How should researchers interpret OR5D16 antibody results in the context of olfactory receptor expression in non-nasal tissues?

Interpreting OR5D16 expression in non-nasal tissues:

• Validation requirements:

  • Employ multiple detection methods (IHC, IF, WB, qPCR) to confirm expression

  • Use highly specific antibodies with minimal cross-reactivity to other olfactory receptors

  • Include appropriate positive and negative tissue controls

• Functional assessment considerations:

  • Investigate potential ligands for OR5D16 in the tissue context

  • Consider downstream signaling mechanisms that may differ from canonical olfactory signal transduction

  • Evaluate co-expression with olfactory signaling components (e.g., G proteins)

• Physiological context:

  • Associate OR5D16 expression with specific cell types and their functions

  • Consider potential roles in tissue-specific processes (e.g., chemosensation, migration, metabolism)

  • Investigate potential associations with pathological conditions

• Comparative analysis:

  • Compare OR5D16 expression patterns with other ectopically expressed olfactory receptors

  • Consider evolutionary conservation of expression in non-nasal tissues across species

  • Evaluate potential functional redundancy within the olfactory receptor family

What strategies can address weak or absent signal when using OR5D16 antibodies?

When facing weak or absent signal with OR5D16 antibodies:

• Sample preparation optimization:

  • Reassess fixation protocols (duration, temperature, fixative composition)

  • Optimize antigen retrieval methods (heat-induced vs. enzymatic, pH conditions)

  • Consider alternative sample preparation methods for membrane proteins

• Antibody-related factors:

  • Titrate antibody concentration beyond recommended ranges (both higher and lower)

  • Evaluate antibody storage conditions and potential degradation

  • Consider alternative OR5D16 antibodies targeting different epitopes

• Detection system enhancement:

  • Implement signal amplification methods (tyramide signal amplification, polymer-based detection)

  • Increase incubation times for primary and secondary antibodies

  • Optimize visualization reagents (substrate concentration, development time)

• Background reduction:

  • Enhance blocking protocols (duration, blocking agent composition)

  • Include additional washing steps and detergents to reduce non-specific binding

  • Consider tissue-specific autofluorescence quenching methods for IF applications

• Biological considerations:

  • Verify OR5D16 expression in your specific samples via orthogonal methods

  • Consider potential epitope masking due to protein interactions or modifications

  • Evaluate expression levels and potential need for enrichment techniques

How can researchers minimize background and non-specific binding when using OR5D16 antibodies?

To minimize background and non-specific binding:

• Blocking optimization:

  • Test different blocking agents (BSA, serum, commercial blockers) at various concentrations

  • Extend blocking time to improve saturation of non-specific binding sites

  • Consider dual blocking strategies (protein block followed by commercial blocker)

• Antibody dilution and incubation:

  • Optimize antibody dilution within and beyond the recommended ranges (1:50-1:200 for IHC, 1:500-1:1000 for WB)

  • Evaluate temperature effects (4°C overnight versus room temperature short-term incubation)

  • Use antibody diluents containing blocking proteins and mild detergents

• Washing protocol refinement:

  • Increase number and duration of wash steps

  • Test different wash buffers (PBS, TBS, with various detergent concentrations)

  • Ensure complete buffer exchanges between washes

• Sample-specific considerations:

  • Pre-absorb sera or antibody solutions with tissues known to cause cross-reactivity

  • Include additives to reduce tissue-specific background (e.g., fish gelatin for fatty tissues)

  • Consider tissue pre-treatments to reduce endogenous enzyme activities or autofluorescence

• Detection system optimization:

  • Select detection systems with minimal cross-reactivity to your sample species

  • Use highly cross-adsorbed secondary antibodies

  • Consider direct labeling approaches to eliminate secondary antibody background

What are the key considerations for quantitative analysis of OR5D16 expression across different experimental platforms?

For robust quantitative analysis of OR5D16 expression:

• Standardization across platforms:

  • Develop consistent scoring systems applicable to multiple detection methods

  • Establish normalization protocols for cross-platform comparison

  • Include calibration standards when possible

• Quantification methods:

  • For IHC/IF: Consider H-score, Allred score, or digital image analysis

  • For WB: Use densitometry with appropriate normalization controls

  • For ELISA: Establish standard curves with recombinant proteins or synthetic peptides

• Statistical considerations:

  • Determine appropriate statistical tests based on data distribution

  • Account for technical and biological variability

  • Consider power analysis to ensure adequate sample sizes

• Validation requirements:

  • Verify key findings with orthogonal detection methods

  • Ensure biological replicates to account for sample heterogeneity

  • Validate quantification methods with samples of known expression levels

• Reporting standards:

  • Document detailed methodological parameters (antibody catalog numbers, dilutions, incubation conditions)

  • Report both positive and negative results with appropriate controls

  • Include representative images showing the full range of expression patterns observed

How should researchers report OR5D16 antibody validation data in publications?

For comprehensive reporting of OR5D16 antibody validation:

• Antibody identification information:

  • Provide complete antibody details: supplier, catalog number, lot number, host species, clonality

  • Specify the immunogen sequence used to generate the antibody (e.g., DVKDAIRKIINTKYFHIKHRHWYPF)

  • Include information on antibody purification method

• Validation experiments:

  • Document specificity testing methods (Western blot, peptide competition, knockdown controls)

  • Report results from tissue panels showing expected versus observed reactivity patterns

  • Include cross-reactivity testing against similar protein family members

• Application-specific validation:

  • Provide optimization parameters for each application (dilutions, incubation conditions)

  • Include positive and negative control data specific to each application

  • Report limitations in specific applications or tissues

• Visual documentation:

  • Include representative images showing specific versus non-specific staining

  • Provide full blot images for Western blot applications

  • Document any discrepancies between expected and observed results

• Protocol transparency:

  • Provide detailed protocols as supplementary information

  • Specify any deviations from manufacturer recommendations

  • Include troubleshooting steps implemented to optimize performance