OR52A4 Antibody

What is OR52A4 and what is its biological function?

OR52A4 (Olfactory Receptor Family 52 Subfamily A Member 4) is a member of the G-protein-coupled receptor (GPCR) family that functions in olfactory sensory perception. These receptors interact with odorant molecules in the nasal epithelium to initiate neuronal responses that trigger smell perception . OR52A4 belongs to the largest gene family in the genome - the olfactory receptor gene family . These receptors share a characteristic 7-transmembrane domain structure with many neurotransmitter and hormone receptors and mediate G protein-coupled signal transduction in response to odorants .

The protein is primarily localized to the cell membrane as a multi-pass membrane protein, consistent with its function in transmembrane signaling . The gene encoding this receptor is intronless (single coding-exon), which is characteristic of the olfactory receptor gene family .

What applications can OR52A4 antibodies be used for in research?

Based on validation data from multiple manufacturers, OR52A4 antibodies have been tested and confirmed for the following applications:

When designing experimental protocols, researchers should perform preliminary optimization experiments to determine the optimal dilution for their specific sample type and detection system .

What is the difference between calculated and observed molecular weights for OR52A4?

An important consideration for researchers using OR52A4 antibodies is the discrepancy between calculated and observed molecular weights:

• Calculated molecular weight: ~34-35 kDa

• Observed molecular weight in Western blots: ~72 kDa

This significant difference may be attributed to several factors:

• Post-translational modifications such as glycosylation, which is common in membrane proteins

• Protein dimerization

• The hydrophobic nature of the transmembrane domains affecting migration in SDS-PAGE

Researchers should be aware of this discrepancy when interpreting Western blot results and plan appropriate controls to confirm specificity .

How do I select the appropriate OR52A4 antibody for my research?

Selection of an OR52A4 antibody should be based on several critical considerations:

• Immunogen specificity: Antibodies generated against different epitopes may yield varying results. The search results indicate that available antibodies target different regions:

  • Epitope region 211-260 amino acids (human OR52A4)

  • Epitope region 194-243 amino acids (human OR52A4)

• Species reactivity: Most commercial antibodies recognize human OR52A4, while some cross-react with mouse and rat proteins . When working with non-human samples, verify species cross-reactivity in the antibody documentation.

• Validation evidence: Request validation data from manufacturers, particularly Western blot images showing the expected band pattern. Some antibodies have published validation data showing detection in specific cell lines such as K562 cells .

• Clonality: Most available OR52A4 antibodies are rabbit polyclonal antibodies . Polyclonal antibodies recognize multiple epitopes and may provide stronger signals but potentially with higher background compared to monoclonal alternatives.

What controls should be included when working with OR52A4 antibodies?

Implementing proper controls is essential for interpreting results with OR52A4 antibodies:

• Positive control: Use tissue or cell lines known to express OR52A4. K562 cells have been documented as expressing detectable levels of OR52A4 .

• Negative control: Include samples from tissues known not to express OR52A4 or use siRNA knockdown of OR52A4 in positive samples.

• Isotype control: Include the appropriate isotype control (rabbit IgG for most commercial OR52A4 antibodies) to distinguish non-specific binding due to the antibody class.

• Blocking peptide control: Some manufacturers offer blocking peptides derived from the immunogen sequence that can be used to confirm antibody specificity . A diminished or absent signal when the antibody is pre-incubated with the blocking peptide indicates specificity.

• Secondary antibody only control: To assess background from the secondary detection system.

What are the optimal conditions for Western blot detection of OR52A4?

For successful Western blot detection of OR52A4, consider these methodological details:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for cell/tissue lysis

  • For membrane proteins like OR52A4, addition of 0.1% SDS can improve solubilization

  • Avoid boiling samples above 70°C to prevent aggregation of transmembrane proteins

• Gel electrophoresis:

  • Use 10-12% polyacrylamide gels for optimal resolution of the 34-72 kDa range

  • Load 20-50 μg of total protein per lane depending on expression levels

• Transfer conditions:

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

  • Use 10-20% methanol in transfer buffer for improved transfer of membrane proteins

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk or BSA in TBST

  • Incubate with primary antibody at recommended dilution (1:500-1:2000) overnight at 4°C

  • Wash thoroughly with TBST (at least 3x10 minutes)

  • Incubate with compatible HRP-conjugated secondary antibody

• Detection:

  • Use enhanced chemiluminescence (ECL) for detection

  • Be prepared to visualize bands at both the calculated (34-35 kDa) and observed (72 kDa) molecular weights

How should I approach immunofluorescence experiments with OR52A4 antibodies?

For optimal immunofluorescence results with OR52A4 antibodies:

• Fixation and permeabilization:

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

  • Permeabilize with 0.1-0.3% Triton X-100 (5-10 minutes) to access intracellular epitopes

  • For membrane proteins like OR52A4, gentler permeabilization with 0.1% saponin may preserve antigenicity better

• Blocking:

  • Block with 5-10% normal serum (from the species of secondary antibody) with 1% BSA

  • Include 0.1% Tween-20 to reduce background

• Antibody incubation:

  • Dilute primary antibody as recommended (1:200-1:1000)

  • Incubate overnight at 4°C in a humidified chamber

  • Use fluorophore-conjugated secondary antibodies at 1:200-1:1000 dilution

• Expected localization pattern:

  • OR52A4 should primarily show cell membrane localization as a multi-pass membrane protein

  • Some perinuclear/ER staining may be observed due to protein synthesis and trafficking

• Counterstaining:

  • Use DAPI for nuclear staining

  • Consider membrane markers (e.g., WGA) to confirm membrane localization

How do I verify the specificity of my OR52A4 antibody?

Verifying antibody specificity is crucial for reliable results:

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • Compare results with and without peptide competition

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

• Genetic approaches:

  • Use siRNA or CRISPR-Cas9 to knock down OR52A4 expression

  • Compare antibody signal in wild-type versus knockdown samples

  • Specific signals should decrease proportionally to knockdown efficiency

• Cross-validation:

  • Compare results using antibodies raised against different epitopes of OR52A4

  • Consistent results with multiple antibodies support specificity

  • Consider using antibodies from different host species or with different clonality

• Correlation with mRNA expression:

  • Correlate protein detection with mRNA levels across different tissues or cell lines

  • Concordance between protein and mRNA abundance supports antibody specificity

What are common issues when working with OR52A4 antibodies and how can they be resolved?

What are the optimal storage conditions for OR52A4 antibodies?

Based on manufacturer recommendations , proper storage is essential for maintaining antibody performance:

• Long-term storage:

  • Store at -20°C for up to one year from the date of receipt

  • Antibodies are typically supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide for stability

• Working storage:

  • For frequent use, store small aliquots at 4°C for up to one month

  • Avoid repeated freeze-thaw cycles which degrade antibody quality

• Handling precautions:

  • Allow antibody to reach room temperature before opening

  • Briefly centrifuge vials before opening to collect liquid at the bottom

  • Handle under sterile conditions to prevent contamination

  • Note that many preparations contain sodium azide, which is toxic and requires proper disposal

Regular quality control testing of older antibody stocks is recommended to ensure continued specificity and sensitivity.

How can OR52A4 antibodies be used in studying olfactory receptor function beyond traditional methods?

Recent advances suggest several innovative applications:

• Olfactory receptor trafficking studies:

  • Using OR52A4 antibodies to track receptor internalization and recycling following ligand exposure

  • Combining with live-cell imaging techniques to monitor dynamic processes

• Proximity ligation assays (PLA):

  • Investigating protein-protein interactions between OR52A4 and associated G proteins

  • Detecting co-localization with other components of olfactory signaling cascades

• ChIP-seq applications:

  • Examining transcription factor binding to OR52A4 promoter regions

  • Investigating epigenetic regulation of OR52A4 expression

• Mass spectrometry validation:

  • Using immunoprecipitation with OR52A4 antibodies followed by mass spectrometry

  • Identifying post-translational modifications and interaction partners

When designing such advanced applications, researchers should thoroughly validate antibody specificity for the particular application and optimize experimental conditions through pilot studies.

How do commercially available OR52A4 antibodies compare in performance and specifications?

When selecting an antibody, researchers should consider which validation data is most relevant to their specific application and experimental system.