OR4A4P/OR4A47 Antibody

What is OR4A4P/OR4A47 antibody and what are its targets?

OR4A4P/OR4A47 antibody is a rabbit polyclonal antibody that specifically recognizes both Putative olfactory receptor 4A4 (OR4A4P) and Olfactory receptor 4A47 (OR4A47) proteins in humans . These target proteins are members of the olfactory receptor family, with OR4A47 functioning as an odorant receptor located in the cell membrane as a multi-pass membrane protein . The antibody is typically generated using synthesized peptides derived from the C-terminal region of the human olfactory receptor proteins, usually targeting amino acids in the 205-233 region for OR4A47 and 250-299 region for OR4A4P .

What applications is this antibody validated for?

The OR4A4P/OR4A47 antibody has been validated for multiple research applications with specific dilution recommendations:

These applications have been confirmed through testing with cell lines including LOVO, HT29, and A549 cells . The antibody's specificity in Western blot analysis has been demonstrated by blocking with synthesized peptide, confirming target-specific binding .

How should the antibody be stored and handled?

Proper storage and handling are crucial for maintaining antibody activity:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles to preserve antibody integrity

• For short-term storage (up to 2 weeks), the antibody can be maintained at 2-8°C

• For long-term storage, keep at -20°C in small aliquots to minimize freeze-thaw cycles

• The antibody is typically supplied in a buffer containing PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide

What is the species reactivity of the OR4A4P/OR4A47 antibody?

Different commercial versions of the antibody show varying species reactivity profiles:

• All versions react with human samples

• Some variants also show reactivity with rat and mouse samples

• Species cross-reactivity should be verified when working with non-human samples, as reactivity may vary between different product offerings

What are the basic physical and biochemical properties of this antibody?

The OR4A4P/OR4A47 antibody has the following characteristics:

• Host/Source: Rabbit

• Clonality: Polyclonal

• Isotype: IgG

• Form: Liquid

• Concentration: Typically 1 mg/mL

• Purification Method: Affinity-purified from rabbit antiserum using epitope-specific immunogen or protein A column followed by peptide affinity purification

• Calculated Molecular Weight of target protein: ~34,760 Da

How can I validate the specificity of OR4A4P/OR4A47 antibody in my experiments?

Validating antibody specificity is critical for reliable research outcomes:

• Peptide competition assay: Use the synthesized immunogenic peptide as a competitive blocker. Western blot analysis has demonstrated that signal detection is blocked when the antibody is pre-incubated with the synthesized peptide, confirming specificity .

• Positive control selection: Use cell lines known to express OR4A4P/OR4A47 such as LOVO, HT29, or NCI-H460 cells, which have been documented to express the target proteins .

• Negative controls: Include samples where the primary antibody is omitted or replaced with normal rabbit IgG at the same concentration.

• Multiple detection methods: Confirm expression using different applications (WB, IF, ICC) to corroborate findings.

• siRNA knockdown: For functional validation, compare antibody signal between wild-type cells and those where the target gene has been silenced.

Western Blot Protocol Optimization

• Sample preparation: Extract proteins from cells using RIPA buffer supplemented with protease inhibitors

• Protein loading: Load 25-35 μg protein per lane (as used with NCI-H460 cells)

• Antibody dilution: Use at 1:1000 dilution for optimal results

• Incubation conditions: Incubate with primary antibody overnight at 4°C

• Detection system: Use HRP-conjugated secondary antibody and ECL detection

• Expected result: A band corresponding to approximately 35 kDa should be visible

Immunofluorescence Protocol Optimization

• Cell fixation: Fix cells with 4% paraformaldehyde for 15 minutes

• Permeabilization: Use 0.2% Triton X-100 for 10 minutes

• Blocking: Block with 3% BSA in PBS for 1 hour

• Primary antibody: Apply at 1:200-1:500 dilution, incubate overnight at 4°C

• Secondary antibody: Use fluorescently-labeled anti-rabbit antibody

• Counterstaining: DAPI for nuclear visualization

• Expected pattern: Membrane and cytoplasmic staining pattern for these transmembrane proteins

How can I incorporate OR4A4P/OR4A47 antibody into antibody nanocage designs?

Antibody nanocages represent an advanced application where antibodies are assembled into precisely oriented structures with controlled valency:

• Compatibility assessment: OR4A4P/OR4A47 antibodies could potentially be incorporated into nanocage designs if constructed as Fc fusion proteins or complete IgG formats .

• Design considerations:

  • The antibody would need to be positioned along the two-fold symmetry axes of the target architecture

  • Fc-binding proteins like protein A can be used to recognize the Fc domain of the IgG constant region

  • Various geometric arrangements (dihedral, tetrahedral, octahedral, or icosahedral) can be considered for nanocage formation

• Assembly method: The assembly would involve:

  • Rigid fusion of Fc-binding proteins to monomeric helical linkers

  • Further fusion to cyclic homo-oligomers

  • The designed cage-forming protein would be a cyclic oligomer with antibody-binding domains

• Stability evaluation: Assembled antibody nanocages should be tested for stability using techniques like size exclusion chromatography (SEC) .

• Potential applications: Such nanocages could enhance binding avidity and receptor clustering for signaling pathway studies .

Weak or No Signal in Western Blot

• Antibody titration: Test different dilutions ranging from 1:500 to 1:2000

• Protein loading: Increase protein amount to 35-50 μg per lane

• Exposure time: Extend exposure time for ECL detection

• Transfer efficiency: Check transfer using a reversible stain like Ponceau S

• Extraction method: Use stronger lysis buffers with denaturation for membrane proteins

• Blocking optimization: Test different blocking agents (BSA vs. milk)

High Background in Immunofluorescence

• Antibody dilution: Use more dilute antibody preparation (1:500-1:1000)

• Blocking enhancement: Extend blocking time to 2 hours or overnight

• Wash optimization: Increase number and duration of washes

• Fixation adjustment: Test different fixation methods (methanol vs. paraformaldehyde)

• Secondary antibody: Ensure proper dilution and minimal cross-reactivity

How can I analyze expression patterns of OR4A4P/OR4A47 in different tissues and disease states?

• Tissue panel analysis: Western blot or immunohistochemistry can be performed on multiple tissue lysates or sections to map expression patterns.

• Cell type identification: Combine OR4A4P/OR4A47 antibody with cell-type-specific markers in co-immunofluorescence to identify which cells express these receptors.

• Disease state comparison:

  • Compare expression in normal vs. diseased tissues

  • Quantify expression levels using densitometry for Western blots

  • Use digital pathology tools for quantifying immunohistochemistry signals

• Subcellular localization: Use confocal microscopy with markers for different cellular compartments to determine precise localization of OR4A4P/OR4A47.

• Expression correlation: Analyze if expression correlates with other markers or clinical outcomes in patient samples.

What are the limitations of using polyclonal antibodies for OR4A4P/OR4A47 research?

While polyclonal antibodies offer advantages in signal amplification, researchers should consider:

• Batch-to-batch variation: Different production lots may have variations in specificity and sensitivity.

• Cross-reactivity concerns: The high sequence similarity between OR4A4P and OR4A47 (and potentially other olfactory receptors) means the antibody recognizes both targets, which could complicate interpretation in systems where differential expression is important.

• Limited supply: Polyclonal antibodies represent a finite resource from each immunized animal.

• Epitope multiplicity: Different antibody molecules in the polyclonal preparation may recognize different epitopes on the target protein.

• Research context: These limitations should be considered when designing experiments requiring high specificity or when developing diagnostic applications.

How can I differentiate between OR4A4P and OR4A47 signals in my experiments?

Distinguishing between the two highly similar proteins requires specialized approaches:

• Complementary molecular techniques: Use RT-qPCR with gene-specific primers to quantify mRNA levels of each target separately.

• Unique peptide targeting: Consider custom antibody development against unique regions of each protein if they exist.

• Genetic manipulation: Use CRISPR/Cas9 or siRNA to specifically knock down one target and observe changes in antibody signal.

• Mass spectrometry: For definitive protein identification, use immunoprecipitation followed by mass spectrometry to identify specific peptides unique to each protein.

• Bioinformatic analysis: Compare genomic data with protein expression to determine if both genes are expressed in your experimental system.