OR5AS1 Antibody

What is OR5AS1 and what cellular functions does it perform?

OR5AS1 (Olfactory Receptor Family 5 Subfamily AS Member 1, also known as Olfactory receptor OR11-168) is a member of the olfactory receptor protein family. These receptors interact with odorant molecules in the nose to initiate neuronal responses that trigger smell perception. OR5AS1 belongs to the large family of G-protein-coupled receptors (GPCRs) that arise from single coding-exon genes. Like other olfactory receptors, OR5AS1 has a 7-transmembrane domain structure similar to many neurotransmitter and hormone receptors and is responsible for the recognition and G protein-mediated transduction of odorant signals. The olfactory receptor gene family is the largest in the human genome, with OR5AS1 being one specific member of this extensive family. The protein plays a crucial role in the molecular mechanisms underlying smell perception and signal transduction pathways .

What types of OR5AS1 antibodies are currently available for research applications?

Current research-grade OR5AS1 antibodies are predominantly polyclonal antibodies produced in rabbits. These antibodies are typically generated by immunizing rabbits with synthesized peptides derived from human OR5AS1, particularly from the internal region spanning amino acids 61-110. Available antibodies are generally unconjugated (not linked to reporter molecules) and purified from rabbit antiserum using affinity chromatography with epitope-specific immunogens. Most commercial preparations have a concentration of approximately 1 mg/ml and are supplied in buffered aqueous solutions containing preservatives such as sodium azide and stabilizers like glycerol. These antibodies are specifically designed for human OR5AS1 detection and are validated for techniques including ELISA and immunofluorescence (IF/ICC) .

What is the molecular characteristics of the OR5AS1 protein?

The OR5AS1 protein is characterized by the following molecular properties:

The protein features the characteristic structure of G-protein coupled receptors with seven transmembrane domains that facilitate signal transduction following odorant binding. OR5AS1 is involved in the molecular mechanisms of olfactory perception, converting chemical stimuli into neuronal responses .

What are the validated applications for OR5AS1 antibodies and their recommended protocols?

OR5AS1 antibodies have been validated for specific research applications with established protocols and recommended dilutions:

For ELISA applications, the high dilution factor (1:5000) indicates high sensitivity for antigen detection. For immunofluorescence applications, researchers should optimize the dilution within the 1:100-1:500 range depending on cell type and fixation method. All antibody applications should include appropriate negative and positive controls to validate specificity. It's important to note that optimal dilutions may vary based on sample type, preparation methods, and detection systems, so researchers should perform titration experiments for their specific experimental conditions .

How should OR5AS1 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of OR5AS1 antibodies is critical for maintaining their specificity and sensitivity in experimental applications. The antibodies should be stored at -20°C for long-term preservation. To minimize freeze-thaw cycles that can degrade antibody quality, it is recommended to aliquot the antibody solution into smaller volumes upon receipt. When working with the antibody, thaw aliquots at 4°C or on ice rather than at room temperature to prevent protein denaturation. Most commercial preparations are supplied in a stabilizing buffer containing PBS (without Mg²⁺ and Ca²⁺) at pH 7.4, with 150 mM NaCl, 0.02% sodium azide, and 50% glycerol to enhance stability. When diluting the antibody for experimental use, use fresh, cold buffer and preferably prepare working dilutions immediately before use. If storing diluted antibody is necessary, keep at 4°C for short periods (1-2 weeks) or re-aliquot and freeze for longer storage .

What cellular models are appropriate for studying OR5AS1 expression and function?

Based on available research data, several cellular models have demonstrated utility for OR5AS1 studies:

When selecting a cellular model, researchers should consider the endogenous expression level of OR5AS1, which varies across tissues and cell types. For functional studies, heterologous expression systems like HEK-293 cells can be transfected with OR5AS1 expression constructs. For studying physiological roles, neuronal cell lines or primary olfactory neurons may provide more relevant contexts. Verification of OR5AS1 expression in the chosen model is recommended through RT-PCR, western blotting, or immunofluorescence before conducting detailed functional studies .

How can researchers distinguish between OR5AS1 and other closely related olfactory receptors in experimental systems?

Distinguishing OR5AS1 from other olfactory receptors presents significant challenges due to sequence homology within this large gene family. Researchers should implement multiple validation strategies:

• Antibody Specificity Verification:

  • Perform peptide competition assays where the immunizing peptide blocks specific binding

  • Test antibody reactivity against recombinant OR5AS1 versus related family members

  • Validate with genetic approaches (siRNA knockdown, CRISPR/Cas9 knockout)

• Molecular Biology Approaches:

  • Use highly specific PCR primers targeting unique regions of OR5AS1 mRNA

  • Employ FISH (Fluorescence In Situ Hybridization) with sequence-specific probes

  • Sequence verification of cloned constructs before expression studies

• Functional Discrimination:

  • Utilize ligand response profiles, as different olfactory receptors respond to distinct odorants

  • Employ G-protein coupling specificity as a distinguishing characteristic

When conducting immunofluorescence studies, validation can be performed using peptide blocking, as demonstrated in the LOVO cell immunofluorescence experiments where the antibody signal was specifically eliminated by pre-incubation with the synthesized peptide immunogen . For genetic manipulation studies, guide RNA design for CRISPR or siRNA should target unique sequences that don't occur in related olfactory receptors. Sequence alignment analysis should precede any experimental design to identify these distinguishing regions .

What are the current limitations in OR5AS1 antibody-based research and how might they be addressed?

Current OR5AS1 antibody research faces several technical and biological limitations:

To address these limitations, researchers might consider developing tissue-specific validation protocols, creating epitope-tagged OR5AS1 expression constructs for antibody validation, or employing alternative detection methods such as RNA-based approaches (RNAscope, RNA-seq) to complement antibody studies. For critical experiments, using multiple antibodies targeting different epitopes can provide stronger evidence of specificity. Advanced approaches like proximity ligation assays could also enhance detection specificity when studying protein-protein interactions involving OR5AS1 .

How can researchers effectively study post-translational modifications of OR5AS1?

Studying post-translational modifications (PTMs) of OR5AS1 requires specialized approaches beyond standard antibody detection:

• PTM-Specific Antibodies:

  • Use antibodies that specifically recognize phosphorylated, glycosylated, or ubiquitinated forms of OR5AS1

  • Validate specificity with phosphatase or deglycosylation enzyme treatments

• Mass Spectrometry Approaches:

  • Immunoprecipitate OR5AS1 followed by MS analysis to identify modification sites

  • Compare PTM profiles under different physiological conditions

• Site-Directed Mutagenesis:

  • Create constructs with mutations at predicted PTM sites

  • Compare functional outcomes between wild-type and mutant proteins

• Inhibitor Studies:

  • Use kinase, glycosyltransferase, or other enzyme inhibitors to modulate PTMs

  • Monitor effects on OR5AS1 function, localization, and turnover

Given that GPCRs like OR5AS1 commonly undergo phosphorylation affecting their signaling and internalization, these modifications likely play crucial roles in regulating olfactory receptor function. Researchers should also consider that standard western blotting may reveal multiple bands representing different modified forms of OR5AS1. When studying these modifications, it's essential to validate findings using multiple approaches, as different experimental techniques may yield complementary information about the PTM landscape of OR5AS1 .

What are common challenges when using OR5AS1 antibodies in immunofluorescence and how can they be resolved?

Researchers commonly encounter several challenges when using OR5AS1 antibodies for immunofluorescence:

Based on the LOVO cell immunofluorescence data, successful OR5AS1 detection requires careful optimization. The peptide competition experiment demonstrated in the LOVO cell study provides an excellent specificity control and should be included when validating staining patterns. Additionally, researchers should optimize fixation methods, as over-fixation can mask epitopes, particularly for membrane proteins like OR5AS1. Permeabilization conditions are also critical—excessive permeabilization may disrupt membrane protein organization, while insufficient permeabilization prevents antibody access to intracellular epitopes .

How can researchers optimize ELISA protocols for maximum sensitivity when detecting OR5AS1?

Optimizing ELISA protocols for OR5AS1 detection requires systematic adjustment of multiple parameters:

• Antibody Dilution Optimization:

  • While 1:5000 is recommended, perform titration experiments (1:1000 to 1:10000)

  • Find optimal primary antibody concentration that maximizes signal-to-noise ratio

• Blocking Optimization:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Optimize blocking time (1-3 hours) and temperature

• Sample Preparation:

  • For cell/tissue lysates, optimize extraction buffers with appropriate detergents

  • Consider membrane protein extraction kits for improved yield

• Detection System Selection:

  • Compare HRP-conjugated vs. alkaline phosphatase secondary antibodies

  • Evaluate chemiluminescent vs. colorimetric substrates for sensitivity needs

• Incubation Parameters:

  • Test different incubation temperatures (4°C, room temperature, 37°C)

  • Evaluate various incubation times (overnight vs. shorter incubations)

When developing sandwich ELISA protocols, use capture and detection antibodies targeting different epitopes of OR5AS1 to improve specificity. Researchers should also consider including recombinant OR5AS1 protein as a positive control to generate standard curves and determine the assay's limit of detection. For challenging samples with low OR5AS1 expression, signal amplification systems or more sensitive detection methods like electrochemiluminescence may be beneficial .

What strategies can improve specificity when detecting OR5AS1 in complex tissue samples?

Detecting OR5AS1 in complex tissue samples presents unique challenges due to potential cross-reactivity with other olfactory receptors and background signals. Several strategies can enhance specificity:

• Pre-absorption Controls:

  • Pre-incubate antibody with excess immunizing peptide before application

  • Include this as a negative control alongside experimental samples

• Dual-labeling Approaches:

  • Co-stain with markers of olfactory neurons or relevant cellular compartments

  • Use confocal microscopy to confirm co-localization patterns

• Signal Amplification with Specificity Checks:

  • Employ tyramide signal amplification while maintaining stringent washing

  • Use proximity ligation assays when studying protein interactions

• Tissue-specific Processing:

  • Optimize fixation protocols specifically for olfactory epithelium

  • Consider antigen retrieval methods optimized for GPCR detection

• Genetic Controls:

  • When possible, use tissue from OR5AS1 knockout models as negative controls

  • Employ tissue with known OR5AS1 overexpression as positive controls

For olfactory epithelium samples specifically, researchers should consider using specialized fixation methods that preserve membrane protein structure while allowing antibody accessibility. Sequential probing with multiple antibodies against different OR5AS1 epitopes can provide additional confidence in the specificity of detection. When using fluorescence detection, autofluorescence reduction techniques may be necessary, particularly in tissues with high lipofuscin content .

How should researchers quantitatively analyze OR5AS1 expression data from immunofluorescence studies?

Quantitative analysis of OR5AS1 immunofluorescence requires rigorous approaches to ensure reliability:

• Image Acquisition Parameters:

  • Maintain consistent exposure settings across all experimental conditions

  • Capture multiple fields (minimum 5-10) per condition for statistical validity

  • Include z-stack imaging for membrane proteins like OR5AS1

• Quantification Methods:

  • Measure mean fluorescence intensity within defined cellular regions

  • Quantify percentage of positive cells in heterogeneous populations

  • Assess co-localization with organelle markers using Pearson's correlation coefficient

• Analysis Software Options:

  • ImageJ/FIJI with appropriate plugins for automated analysis

  • CellProfiler for pipeline-based high-throughput quantification

  • Commercial software packages with advanced co-localization analysis

• Statistical Approach:

  • Use appropriate statistical tests based on data distribution

  • Account for cell-to-cell variability with sufficient sample sizes

  • Consider hierarchical statistical models for nested data structures

When analyzing OR5AS1 immunofluorescence in LOVO cells or other models, membrane localization patterns should be distinguished from cytoplasmic signals. For GPCRs like OR5AS1, trafficking between membrane and intracellular compartments may be physiologically relevant. Researchers should therefore consider subcellular distribution patterns in their analysis, potentially quantifying the ratio of membrane to cytoplasmic signal as a measure of receptor trafficking .

What are appropriate controls for validating OR5AS1 antibody specificity in research applications?

Comprehensive validation of OR5AS1 antibody specificity requires multiple control strategies:

The peptide competition control demonstrated in the LOVO cell immunofluorescence experiment is particularly valuable, showing that the antibody signal can be specifically blocked by the synthetic peptide used for immunization. This control directly assesses the epitope specificity of the antibody. For optimal validation, researchers should implement multiple control types rather than relying on a single approach. When publishing OR5AS1 research, detailed documentation of these validation experiments should be included to establish credibility of the findings .

How can researchers integrate OR5AS1 antibody data with other omics approaches for comprehensive functional analysis?

Integrative multi-omics approaches provide powerful insights into OR5AS1 function beyond what antibody-based methods alone can offer:

• Transcriptomics Integration:

  • Correlate protein expression (antibody-based) with mRNA levels (RNA-seq)

  • Identify regulatory networks affecting OR5AS1 expression

  • Use single-cell RNA-seq to characterize cell-specific expression patterns

• Proteomics Complementation:

  • Compare antibody-based detection with mass spectrometry data

  • Identify protein interaction partners through IP-MS approaches

  • Characterize post-translational modifications affecting function

• Functional Genomics Correlation:

  • Link CRISPR/Cas9 or RNAi phenotypes with protein expression patterns

  • Correlate genomic variants (SNPs) with protein expression/function

• Structural Biology Integration:

  • Combine localization data with structural predictions/models

  • Use antibody epitope information to validate structural features

• Computational Analysis Framework:

  • Develop integrated visualization tools for multi-omics data

  • Apply machine learning to identify patterns across datasets

  • Use pathway analysis to place OR5AS1 in broader biological context

When integrating antibody-based data with other approaches, researchers should be mindful of the different sensitivities and specificities of each method. Apparent discrepancies between techniques may reflect biological realities rather than technical failures. For example, post-translational modifications might affect antibody recognition without altering mRNA levels, or protein stability differences could cause protein and mRNA levels to diverge. A multi-omics approach provides the most comprehensive understanding of OR5AS1 biology by leveraging the strengths of complementary techniques .

What emerging technologies might enhance OR5AS1 detection and functional characterization?

Several cutting-edge technologies hold promise for advancing OR5AS1 research:

• Advanced Imaging Approaches:

  • Super-resolution microscopy (STORM, PALM) for nanoscale localization

  • Live-cell imaging with genetically encoded tags to study trafficking dynamics

  • Expansion microscopy for improved visualization of subcellular distribution

• Antibody Engineering:

  • Nanobodies or single-domain antibodies for improved access to conformational epitopes

  • Bispecific antibodies targeting OR5AS1 and interaction partners simultaneously

  • Recombinant antibody fragments with enhanced tissue penetration

• Functional Assays:

  • CRISPR activation/inhibition systems for precise modulation of OR5AS1 expression

  • Biosensor development for real-time monitoring of OR5AS1 activation

  • Microfluidic systems for high-throughput ligand screening

• Structural Approaches:

  • Cryo-EM studies of OR5AS1 structure in different conformational states

  • Hydrogen-deuterium exchange mass spectrometry for dynamics analysis

  • Computational modeling informed by antibody epitope mapping

These emerging technologies will likely enable researchers to move beyond static detection of OR5AS1 toward understanding its dynamic behavior in physiological contexts. For example, combining traditional antibody approaches with newer technologies like proximity labeling (BioID, APEX) could reveal the OR5AS1 interactome in specific cellular compartments. Similarly, integrating antibody detection with functional calcium imaging could link receptor expression patterns to odorant response properties in sensory neurons .

How might understanding OR5AS1 contribute to broader knowledge about olfactory receptor biology?

OR5AS1 research has potential to inform multiple aspects of olfactory receptor biology:

• Receptor Trafficking Mechanisms:

  • Elucidating how OR5AS1 is transported to dendritic cilia of olfactory neurons

  • Understanding quality control mechanisms for GPCRs in the secretory pathway

• Signal Transduction Pathways:

  • Characterizing OR5AS1-specific G-protein coupling preferences

  • Identifying downstream effectors and second messenger systems

• Ligand Selectivity Principles:

  • Determining structural features of OR5AS1 that confer odor specificity

  • Developing structure-activity models applicable to other olfactory receptors

• Evolutionary Relationships:

  • Comparative analysis of OR5AS1 across species to identify conserved functions

  • Understanding subfamily-specific features within the larger olfactory receptor family

• Developmental Regulation:

  • Examining the temporal and spatial expression patterns during olfactory system development

  • Identifying transcriptional regulators controlling OR5AS1 expression

By serving as a model olfactory receptor, OR5AS1 research can provide insights applicable to the broader family of over 400 olfactory receptors in humans. The principles of ligand recognition, signal transduction, and receptor regulation uncovered through OR5AS1 studies may reveal common mechanisms operating across this large gene family. Additionally, understanding OR5AS1 function may contribute to knowledge about the unique "one receptor-one neuron" rule in olfactory system organization and the mechanisms of axon guidance in the formation of the olfactory map .

What are the most promising therapeutic or diagnostic applications emerging from OR5AS1 research?

While OR5AS1 research is primarily fundamental in nature, several translational opportunities may emerge:

• Olfactory Dysfunction Diagnostics:

  • Development of antibody-based assays to assess OR5AS1 expression in olfactory biopsies

  • Correlation of receptor expression patterns with specific anosmia phenotypes

• Olfactory System Development:

  • Understanding OR5AS1's role in the formation of olfactory circuits

  • Potential relevance to developmental disorders affecting sensory systems

• Beyond Olfaction Applications:

  • Investigating potential ectopic expression in non-olfactory tissues

  • Exploring roles in other cellular processes beyond odor detection

• Biomarker Potential:

  • Evaluating OR5AS1 expression changes in neurodegenerative conditions

  • Assessing possible roles in neuroinflammatory processes

• Therapeutic Target Assessment:

  • Screening for compounds that modulate OR5AS1 activity

  • Exploring potential role in appetite regulation or other physiological processes

While direct therapeutic applications remain speculative, the fundamental knowledge gained about GPCR biology through OR5AS1 research may have broader implications. The understanding of receptor trafficking, quality control mechanisms, and ligand interaction principles could inform drug development for other GPCR targets. Additionally, as olfactory dysfunction is associated with several neurodegenerative conditions (including Parkinson's and Alzheimer's diseases), understanding olfactory receptor biology may contribute to early diagnostic approaches for these disorders .

What are the key considerations for researchers planning to use OR5AS1 antibodies in their work?

Researchers considering OR5AS1 antibody use should prioritize the following considerations:

• Experimental Planning:

  • Clearly define research questions that antibody detection can address

  • Select appropriate positive and negative controls based on OR5AS1 expression patterns

  • Plan comprehensive validation experiments before proceeding to main studies

• Antibody Selection:

  • Choose antibodies validated for your specific application (ELISA, IF/ICC)

  • Consider the epitope location and how it might affect detection in different contexts

  • Review literature for successful use of specific antibody clones/catalog numbers

• Protocol Optimization:

  • Invest time in optimizing dilutions (1:100-1:500 for IF, 1:5000 for ELISA)

  • Adapt fixation and permeabilization protocols for membrane protein detection

  • Include epitope-blocking controls to confirm specificity

• Data Interpretation:

  • Consider potential cross-reactivity with related olfactory receptors

  • Interpret localization patterns in context of OR5AS1's GPCR biology

  • Supplement antibody data with other detection/functional approaches

Researchers should approach OR5AS1 studies with realistic expectations about the challenges of studying individual members of large gene families. The available antibodies require careful validation and optimization, but when properly used, can provide valuable insights into OR5AS1 biology. Integration with complementary approaches (molecular, genetic, functional) will yield the most comprehensive understanding .

What alternative approaches can complement antibody-based detection of OR5AS1?

Several complementary approaches can provide additional insights when used alongside antibody detection:

Combining antibody detection with these complementary approaches provides multiple lines of evidence for OR5AS1 expression and function. For example, validating antibody staining patterns with RNAscope in situ hybridization in the same tissue sections can confirm specificity. Similarly, comparing endogenous OR5AS1 detection with epitope-tagged versions can help validate antibody performance. Researchers should select complementary approaches based on their specific research questions and available resources .

What best practices should researchers follow when publishing data generated using OR5AS1 antibodies?

To ensure reproducibility and reliability of published OR5AS1 antibody data, researchers should adhere to these best practices:

• Comprehensive Antibody Reporting:

  • Provide complete antibody information (manufacturer, catalog number, lot number, RRID)

  • Describe validation experiments performed (Western blot, peptide competition, knockdown)

  • Include images of validation experiments alongside main results

• Detailed Methodological Documentation:

  • Report complete protocols including fixation, blocking, antibody dilutions, and incubation times

  • Specify image acquisition parameters (exposure times, gain settings, microscope details)

  • Describe quantification methods with sufficient detail for replication

• Control Implementation:

  • Include appropriate positive and negative controls in all experiments

  • Show representative images of controls alongside experimental data

  • Quantify control results when presenting quantitative analyses

• Data Presentation Standards:

  • Present uncropped blots/images (as main figures or supplementary material)

  • Avoid excessive image processing that might alter data interpretation

  • Include scale bars on all microscopy images

• Results Interpretation:

  • Discuss antibody limitations and potential cross-reactivity issues

  • Consider alternative explanations for observed patterns

  • Place findings in context of other detection methods when available