OR5L1/OR5L2 Antibody

What are OR5L1 and OR5L2, and why are they targeted with antibodies?

OR5L1 and OR5L2 are members of the olfactory receptor family 5, subfamily L. They belong to the larger G protein-coupled receptor superfamily that plays crucial roles in olfactory signal transduction. These olfactory receptors are primarily expressed in the nasal epithelium but have also been detected in other tissues, suggesting potential non-olfactory functions. Researchers target these proteins with antibodies to study their expression patterns, localization, and potential roles beyond olfaction. The development of specific antibodies against these receptors allows for their detection and characterization in various experimental settings, including tissue samples and cell cultures. This approach is particularly valuable because direct functional studies of olfactory receptors can be challenging due to their specialized nature and expression profiles.

What types of OR5L1/OR5L2 antibodies are currently available for research?

Current research tools include both specific OR5L2 antibodies and dual-specificity OR5L1/2 antibodies. The OR5L2-specific antibody (ABIN655018) is a rabbit polyclonal antibody targeting amino acids 62-90 in the N-terminal region of human OR5L2 . This unconjugated antibody has been validated for Western Blotting (WB) and Immunohistochemistry on paraffin-embedded sections (IHC-P) . For researchers requiring broader detection capability, the OR5L1/2 antibody (DF10254) is a rabbit polyclonal antibody that recognizes both OR5L1 and OR5L2 proteins, with demonstrated reactivity across human, mouse, and rat samples . This antibody has been validated primarily for Western Blotting applications . Both antibodies are generated in rabbits and purified through protein A columns, with the OR5L2-specific antibody undergoing additional peptide affinity purification . Various conjugated forms of the OR5L2 antibody are also available, including APC, biotin, FITC, PE, and HRP conjugates, expanding the range of potential experimental applications .

How do OR5L1-specific, OR5L2-specific, and dual-specificity antibodies differ in terms of research applications?

The choice between specific and dual-specificity antibodies significantly impacts experimental design and data interpretation. OR5L2-specific antibodies like ABIN655018 provide targeted detection of human OR5L2 protein, making them ideal for studies focused exclusively on this receptor subtype . These antibodies are particularly valuable when distinguishing between closely related olfactory receptors in human samples. The specificity is achieved through immunization with a synthetic peptide corresponding to amino acids 62-90 of the human OR5L2 N-terminal region .

What are the optimal conditions for using OR5L1/OR5L2 antibodies in Western blotting experiments?

When using OR5L1/OR5L2 antibodies for Western blotting, several methodological considerations must be addressed for optimal results. Based on the characteristics of available antibodies and standard immunoblotting protocols:

For the OR5L2-specific antibody (ABIN655018), optimal Western blotting conditions include:

• Sample preparation: Tissues or cells should be lysed in a buffer containing protease inhibitors to prevent degradation of the target protein.

• Loading amount: 20-50 μg of total protein per lane is typically sufficient for detection.

• Protein separation: Use 10-12% SDS-PAGE gels, as the calculated molecular weight of OR5L2 is approximately 35 kDa.

• Transfer conditions: Semi-dry or wet transfer to PVDF membranes at 100V for 60-90 minutes.

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody dilution: 1:500 to 1:2000 in blocking buffer, incubated overnight at 4°C.

• Secondary antibody: Anti-rabbit HRP-conjugated antibody at 1:5000 dilution.

• Detection: Enhanced chemiluminescence (ECL) system.

For the dual-specificity OR5L1/2 antibody (DF10254), similar conditions apply, with particular attention to the expected molecular weight of approximately 35 kDa for both target proteins . When using this antibody across different species (human, mouse, rat), additional optimization may be necessary to account for species-specific differences in protein expression levels and potential cross-reactivity.

For both antibodies, proper controls are essential, including positive controls (tissues known to express the target protein), negative controls (tissues not expressing the target), and loading controls (housekeeping proteins like β-actin or GAPDH) to ensure equal protein loading across samples.

How should researchers design immunohistochemistry experiments using OR5L1/OR5L2 antibodies?

Designing robust immunohistochemistry (IHC) experiments with OR5L1/OR5L2 antibodies requires careful consideration of tissue preparation, antigen retrieval, and detection methods. The OR5L2-specific antibody (ABIN655018) has been specifically validated for IHC on paraffin-embedded sections , making it suitable for this application.

A methodological approach for IHC using this antibody includes:

• Tissue preparation:

  • Fix tissues in 10% neutral-buffered formalin for 24-48 hours

  • Process and embed in paraffin

  • Section at 4-6 μm thickness onto positively charged slides

• Antigen retrieval:

  • Deparaffinize and rehydrate sections

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Optimize retrieval conditions (temperature, duration) for specific tissues

• Immunostaining:

  • Block endogenous peroxidase activity with 3% H₂O₂

  • Apply protein block (5% normal goat serum)

  • Incubate with primary antibody at 1:100 to 1:500 dilution overnight at 4°C

  • Apply appropriate HRP-conjugated secondary antibody

  • Develop with DAB substrate and counterstain with hematoxylin

• Controls:

  • Include positive control tissues (olfactory epithelium)

  • Include negative controls (primary antibody omission)

  • Consider peptide competition controls to verify specificity

For the dual-specificity OR5L1/2 antibody (DF10254), additional validation may be necessary before use in IHC, as Western blotting is its primary validated application . Researchers should perform preliminary validation studies to confirm its suitability for IHC across different species.

What sample preparation techniques are recommended for optimal OR5L1/OR5L2 antibody performance?

Sample preparation significantly impacts antibody performance and result reliability. For both OR5L1/OR5L2 antibodies, the following preparation techniques are recommended based on application:

For protein extraction and Western blotting:

• Use fresh or properly stored frozen tissues/cells

• Extract proteins in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 8.0) supplemented with protease inhibitors

• Homogenize tissues thoroughly using mechanical disruption (for tissues) or direct lysis (for cultured cells)

• Clarify lysates by centrifugation (14,000 × g, 15 minutes, 4°C)

• Quantify protein concentration using BCA or Bradford assay

• Store protein extracts at -80°C in single-use aliquots to avoid freeze-thaw cycles

For immunohistochemistry and immunofluorescence:

• Fix tissues immediately after collection (10% neutral-buffered formalin for standard IHC or 4% paraformaldehyde for immunofluorescence)

• Limit fixation time to 24-48 hours to prevent excessive protein cross-linking

• Process tissues carefully to maintain morphology

• Prepare sections at appropriate thickness (4-6 μm for bright-field IHC, 8-10 μm for immunofluorescence)

• Store unstained sections at room temperature or 4°C (short-term) or -20°C (long-term) with desiccant

For both applications, researchers should consider the specific characteristics of olfactory tissues, which can be particularly delicate and prone to autolysis. Rapid processing and careful handling are essential to preserve antigen integrity and morphological features, especially when working with nasal epithelium samples where OR5L1 and OR5L2 are predominantly expressed.

How should researchers determine the analytical measuring interval for OR5L1/OR5L2 antibodies?

Determining the analytical measuring interval (AMI) for OR5L1/OR5L2 antibodies is critical for establishing the range within which reliable quantitative results can be obtained. Following CLSI guidelines, this process involves determining the limit of blank (LOB), limit of detection (LOD), and limit of quantification (LOQ) .

A methodological approach adapted from serology testing validation includes:

• Determination of Limit of Blank (LOB):

  • Test at least 5 blank samples (tissues/cells known not to express OR5L1/OR5L2)

  • Run samples in duplicate over multiple days (≥3) using at least two reagent lots

  • Calculate LOB using parametric or non-parametric methods as described in CLSI EP17-A2

  • Example calculation: Rank all blank measurements from low to high; LOB = value at rank position 0.5 + (n × 0.95)

• Determination of Limit of Detection (LOD):

  • Prepare low-level samples by diluting positive controls in negative matrix

  • Test at least 5 low-level samples in triplicate over multiple days using at least two reagent lots

  • Calculate LOD using the formula: LOD = LOB + (c_p × SD_L)

  • Where c_p is the multiplier for the desired confidence level and SD_L is the standard deviation of low-level samples

• Determination of Limit of Quantification (LOQ):

  • Test samples with progressively higher concentrations of target

  • Identify the lowest concentration at which the coefficient of variation (CV) is ≤20%

  • Verify through additional testing that this concentration consistently yields acceptable precision

This experimental design requires careful preparation of standardized samples and rigorous statistical analysis. For OR5L1/OR5L2 antibodies, researchers should develop standard samples using recombinant proteins or well-characterized cell lines with controlled expression levels of the target proteins.

What approaches should be used to validate the specificity of OR5L1/OR5L2 antibodies?

Validating antibody specificity is crucial, particularly for closely related proteins like OR5L1 and OR5L2. Multiple complementary approaches should be employed to ensure confidence in experimental results:

• Peptide Competition Assays:

  • Pre-incubate the antibody with excess immunizing peptide (for OR5L2 antibody, the peptide corresponding to amino acids 62-90 of the N-terminal region)

  • Run parallel Western blots or IHC with competed and non-competed antibody

  • The specific signal should be significantly reduced or eliminated in the competed samples

• Genetic Models:

  • Test antibody on samples from knockout models (if available)

  • Use siRNA/shRNA knockdown in cell culture models

  • Specificity is confirmed if signal decreases proportionally to knockdown efficiency

• Orthogonal Detection Methods:

  • Compare protein detection with mRNA expression using RT-PCR or RNA-seq

  • Use mass spectrometry to confirm the identity of the detected protein band

  • Employ multiple antibodies targeting different epitopes of the same protein

• Cross-reactivity Testing:

  • Test against recombinant OR5L1 and OR5L2 proteins separately

  • Include related olfactory receptor family members as controls

  • For the dual-specificity antibody, quantify relative affinity for each target

• Cell Line Validation:

  • Test antibodies on cell lines with known expression profiles

  • Use overexpression systems as positive controls

  • Include cells not expressing the target as negative controls

For the OR5L2-specific antibody (ABIN655018), validation should focus on demonstrating negligible cross-reactivity with OR5L1, despite their sequence homology . For the dual-specificity OR5L1/2 antibody (DF10254), validation should quantify the relative binding affinity for each target and demonstrate consistent detection across the claimed species reactivity range (human, mouse, rat) .

How can researchers assess the precision and repeatability of experiments using OR5L1/OR5L2 antibodies?

Assessing precision and repeatability is essential for establishing the reliability of quantitative measurements using OR5L1/OR5L2 antibodies. Adapting the CLSI EP05-A3 guidelines used in serology testing , researchers should evaluate:

• Within-run Repeatability:

  • Test samples with negative, low, medium, and high levels of target protein

  • Analyze each sample in multiple replicates (≥3) within the same experimental run

  • Calculate coefficient of variation (CV) for each concentration level

  • Acceptable repeatability: CV ≤20% for medium and high samples, CV ≤25% for low samples

• Between-run Precision:

  • Test the same sample set across multiple days (≥20 recommended)

  • Use at least two operators and three reagent lots if possible

  • Calculate total precision using variance component analysis

  • Example from serology testing showed total precision of 13.5-17.6% for medium positive samples

• Linearity Assessment:

  • Create a dilution series by serial dilution of high-concentration samples

  • Test each dilution in triplicate

  • Plot observed vs. expected values and calculate percent deviation from linearity

  • Acceptable linearity: ≤20% deviation from expected values

A sample experimental design table for precision assessment of OR5L1/OR5L2 antibodies is shown below:

This rigorous approach allows researchers to establish confidence intervals for their measurements and determine the minimum reliable quantification threshold for their specific experimental setup.

How can OR5L1/OR5L2 antibodies be used in multiplexed imaging applications?

Multiplexed imaging with OR5L1/OR5L2 antibodies enables simultaneous visualization of these receptors alongside other markers, providing spatial context and co-expression information. Advanced methodological approaches include:

• Multi-color Immunofluorescence:

  • Utilize conjugated versions of the OR5L2 antibody (FITC, PE, APC)

  • Combine with antibodies against other proteins of interest raised in different host species

  • Apply sequential staining protocols for antibodies from the same host species

  • Use spectral unmixing to resolve overlapping fluorophore signals

  • Include appropriate controls for each fluorophore and compensation controls

• Tyramide Signal Amplification (TSA):

  • Employ HRP-conjugated OR5L2 antibody with tyramide-fluorophore substrates

  • Use sequential TSA for multiple targets with antibody stripping between rounds

  • This approach enables use of multiple antibodies from the same species

  • Particularly valuable for low-abundance targets like olfactory receptors

• Multiplexed Ion Beam Imaging (MIBI):

  • Conjugate OR5L1/OR5L2 antibodies to isotopically pure metals

  • Combine with other metal-tagged antibodies

  • Analyze using time-of-flight mass spectrometry

  • Enables simultaneous detection of 40+ targets on a single tissue section

• Cyclic Immunofluorescence (CycIF):

  • Apply OR5L1/OR5L2 antibodies in sequential staining rounds

  • Image after each round

  • Chemically inactivate fluorophores between rounds

  • Enables detection of 30+ markers on the same sample

These multiplexed approaches allow researchers to investigate the relationship between OR5L1/OR5L2 expression and other cellular markers, potentially revealing previously unknown associations and functions beyond the olfactory system. The choice of method depends on the specific research question, available instrumentation, and required spatial resolution.

What are the approaches for detecting post-translational modifications of OR5L1/OR5L2 proteins?

Investigating post-translational modifications (PTMs) of OR5L1/OR5L2 proteins requires specialized antibodies and techniques beyond standard detection methods. Currently available antibodies target specific peptide regions but are not designed to detect PTMs . Researchers interested in PTMs should consider:

• Complementary Enrichment Strategies:

  • Immunoprecipitate OR5L1/OR5L2 using available antibodies

  • Analyze precipitated proteins using:

    • Phospho-specific staining (Pro-Q Diamond)

    • Glycoprotein staining (Pro-Q Emerald)

    • Ubiquitin/SUMO blotting with specific antibodies

  • Confirm modifications using mass spectrometry

• Two-dimensional Gel Electrophoresis:

  • Separate proteins by isoelectric point and molecular weight

  • Use available OR5L1/OR5L2 antibodies for Western blotting

  • Identify charge variants indicating potential phosphorylation or other modifications

  • Extract spots for mass spectrometry validation

• Mass Spectrometry-based Approaches:

  • Enrich for OR5L1/OR5L2 proteins through immunoprecipitation

  • Digest with multiple proteases to maximize sequence coverage

  • Apply targeted mass spectrometry approaches:

    • Multiple reaction monitoring (MRM)

    • Parallel reaction monitoring (PRM)

  • Analyze data with PTM-specific search algorithms

• Site-specific Phosphorylation Analysis:

  • Use phosphorylation site prediction tools to identify potential sites

  • Generate custom phospho-specific antibodies against predicted sites

  • Validate using phosphatase treatment controls

  • Confirm functional significance through site-directed mutagenesis

G protein-coupled receptors like OR5L1/OR5L2 commonly undergo multiple PTMs, including phosphorylation, palmitoylation, and glycosylation, which regulate their trafficking, signaling, and desensitization. Understanding these modifications is critical for elucidating the receptors' functional dynamics and could reveal novel therapeutic targets in disorders involving olfactory dysfunction.

How can researchers investigate OR5L1/OR5L2 protein-protein interactions using available antibodies?

Investigating protein-protein interactions (PPIs) of OR5L1/OR5L2 requires leveraging the available antibodies in specific methodological approaches. Researchers can employ:

• Co-immunoprecipitation (Co-IP):

  • Use OR5L2 antibody (ABIN655018) or OR5L1/2 antibody (DF10254) for immunoprecipitation

  • Optimize lysis conditions to preserve membrane protein interactions:

    • Use mild detergents (DDM, CHAPS, or digitonin at 0.5-1%)

    • Include protease and phosphatase inhibitors

    • Maintain physiological pH and salt concentration

  • Perform IP in forward and reverse directions (target interaction partner antibody)

  • Control for non-specific binding using IgG from same species

  • Analyze precipitated complexes by Western blotting or mass spectrometry

• Proximity Ligation Assay (PLA):

  • Combine OR5L1/OR5L2 antibodies with antibodies against suspected interaction partners

  • Use oligonucleotide-labeled secondary antibodies

  • Generate fluorescent signal only when proteins are within 40 nm proximity

  • Analyze using fluorescence microscopy

  • Quantify interaction sites per cell

• FRET/BRET Analysis:

  • Express OR5L1/OR5L2 as fusion proteins with fluorescent/luminescent tags

  • Verify proper expression and localization using available antibodies

  • Measure energy transfer as indication of protein proximity

  • Validate interactions by co-immunoprecipitation with the antibodies

• Crosslinking Mass Spectrometry:

  • Apply membrane-permeable crosslinkers to intact cells expressing OR5L1/OR5L2

  • Immunoprecipitate using available antibodies

  • Analyze crosslinked peptides by mass spectrometry

  • Identify interaction partners and interaction sites

Since OR5L1 and OR5L2 are olfactory receptors (members of the GPCR family), potential interaction partners include G proteins (Gαolf), receptor activity-modifying proteins (RAMPs), receptor transport proteins (RTPs), and various scaffolding proteins. Understanding these interactions could reveal mechanisms regulating receptor trafficking, signaling, and desensitization, potentially uncovering new therapeutic targets for olfactory disorders.

How should researchers address contradictory results when using different OR5L1/OR5L2 antibodies?

When faced with contradictory results using different OR5L1/OR5L2 antibodies, researchers should implement a systematic troubleshooting approach:

• Epitope Analysis:

  • Compare target epitopes of different antibodies

  • The OR5L2-specific antibody (ABIN655018) targets amino acids 62-90 in the N-terminal region

  • The dual-specificity OR5L1/2 antibody (DF10254) may target a different epitope

  • Different epitopes may be differentially accessible due to:

    • Protein conformation changes

    • Post-translational modifications

    • Protein-protein interactions

    • Fixation effects (for IHC)

• Validation Comparison:

  • Review validation data for each antibody

  • Perform side-by-side validation using:

    • Peptide competition assays

    • Knockout/knockdown controls

    • Overexpression systems

  • Document differences in specificity and sensitivity

• Methodological Investigation:

  • Test whether contradictions are method-dependent

  • Compare results across multiple techniques:

    • Western blotting under reducing and non-reducing conditions

    • IHC with different fixation and antigen retrieval methods

    • Flow cytometry with different permeabilization protocols

  • Optimize protocols for each antibody independently

• Isoform/Variant Detection:

  • Investigate whether contradictions might result from detection of different:

    • Splice variants

    • Protein isoforms

    • Post-translationally modified forms

  • Confirm using mass spectrometry or RT-PCR for transcript variants

• Reconciliation Strategy:

  • Triangulate results using a third method (e.g., mass spectrometry, RNA-seq)

  • Consider using both antibodies in parallel with appropriate controls

  • Report contradictory findings transparently in publications

  • Discuss potential biological explanations for discrepancies

When reporting contradictory results, researchers should explicitly document all experimental conditions, antibody details (catalog number, lot, dilution), and validation steps performed. This transparency enables other researchers to interpret the findings appropriately and potentially resolve the contradictions in future studies.

What statistical approaches are recommended for analyzing quantitative data generated using OR5L1/OR5L2 antibodies?

Quantitative analysis of OR5L1/OR5L2 antibody data requires appropriate statistical methods based on experimental design and data characteristics:

• Normalization Strategies:

  • For Western blotting:

    • Normalize to loading controls (β-actin, GAPDH)

    • Consider normalization to total protein (Ponceau S, REVERT)

    • Express results as fold-change relative to control

  • For IHC/IF:

    • Normalize to tissue area or cell count

    • Use internal reference structures when available

    • Consider normalization to background in negative control regions

• Appropriate Statistical Tests:

  • For comparing two groups:

    • Student's t-test (parametric) for normally distributed data

    • Mann-Whitney U test (non-parametric) for non-normal distributions

  • For multiple groups:

    • One-way ANOVA with post-hoc tests (Tukey, Bonferroni) for parametric data

    • Kruskal-Wallis with post-hoc tests for non-parametric data

  • For repeated measures:

    • Repeated measures ANOVA

    • Linear mixed models for complex designs

• Sample Size Determination:

  • Conduct power analysis based on preliminary data

  • Consider variability observed in validation studies

  • Example calculation based on precision data:

    • If CV = 15% (similar to what was observed in serology testing )

    • To detect a 30% difference between groups

    • With α = 0.05 and power (1-β) = 0.8

    • Minimum n = 8 samples per group would be required

• Addressing Variability:

  • Account for batch effects using:

    • Balanced experimental design

    • Inclusion of technical replicates

    • Statistical correction methods (e.g., ANOVA with batch as factor)

  • Handle outliers through:

    • Predefined exclusion criteria

    • Robust statistical methods

    • Non-parametric approaches

• Advanced Analysis Approaches:

  • For complex experiments:

    • Principal component analysis (PCA)

    • Hierarchical clustering

    • Regression models with multiple variables

  • For correlative studies:

    • Pearson or Spearman correlation depending on data distribution

    • Multiple regression for complex relationships

When reporting results, researchers should clearly state the statistical methods used, including software packages, version numbers, and specific parameters. Transparency about data transformations (e.g., log transformation) and outlier handling is essential for reproducibility.

How can researchers distinguish between true OR5L1/OR5L2 signals and background or non-specific binding?

Distinguishing specific OR5L1/OR5L2 signals from background requires rigorous controls and analytical approaches:

• Critical Control Experiments:

  • Negative controls:

    • Primary antibody omission

    • Isotype control antibody (same species, same concentration)

    • Pre-immune serum (if available)

  • Specificity controls:

    • Peptide competition/absorption (using the immunizing peptide)

    • Tissues/cells known to be negative for OR5L1/OR5L2 expression

    • Knockdown/knockout samples (if available)

  • Positive controls:

    • Olfactory epithelium (natural expression)

    • Overexpression systems

    • Recombinant protein standards

• Optimizing Signal-to-Noise Ratio:

  • For Western blotting:

    • Optimize blocking conditions (5% milk vs. BSA)

    • Determine optimal antibody concentration through titration

    • Use high-sensitivity, low-background detection systems

    • Consider longer exposure times with reduced antibody concentration

  • For IHC/IF:

    • Block endogenous peroxidase and biotin

    • Use specialized blocking reagents for neuronal tissues

    • Optimize antigen retrieval methods

    • Employ tyramide signal amplification for low-abundance targets

• Analytical Approaches:

  • Quantify signal intensity relative to background

  • Establish signal threshold based on negative controls

  • Calculate signal-to-noise ratio (SNR)

  • Consider as positive only signals exceeding:

    • Mean background + 3 standard deviations, or

    • 2-3 times background intensity

• Cross-validation Strategies:

  • Compare protein detection with mRNA expression data

  • Use multiple antibodies targeting different epitopes

  • Employ orthogonal detection methods (mass spectrometry)

  • Correlate results across different experimental techniques

• Addressing Common Sources of Non-specific Binding:

  • For neuronal/olfactory tissues:

    • Block lipofuscin autofluorescence (Sudan Black B)

    • Quench tissue autofluorescence (sodium borohydride)

    • Use spectral unmixing to separate autofluorescence

  • For membrane proteins like ORs:

    • Optimize detergent type and concentration

    • Consider native vs. denaturing conditions

    • Be aware of hydrophobic interactions causing non-specific binding

By implementing these strategies, researchers can confidently distinguish true OR5L1/OR5L2 signals from artifacts, ensuring reliable and reproducible experimental outcomes.