OR4K17 Antibody

What is OR4K17 and why is it studied in research?

OR4K17 (also known as olfactory receptor 4K17 or OR14-29) is a member of the G-protein coupled receptor 1 family that functions as an odorant receptor. It is a 315 amino acid multi-pass membrane protein that binds odor molecules and initiates signal transduction cascades leading to the perception of smell . OR4K17 belongs to the largest gene family in the human genome - the olfactory receptor family. Research on this receptor contributes to our understanding of olfactory signal transduction, neuronal development, and sensory processing. The study of OR4K17 may also provide insights into disorders related to olfactory dysfunction and broader neurological conditions.

What types of OR4K17 antibodies are currently available for research?

Several validated polyclonal antibodies against OR4K17 are commercially available. These include rabbit-derived polyclonal antibodies such as:

• Affinity isolated antibodies from Sigma Aldrich (SAB4501711)

• Rabbit polyclonal antibodies from Antibodies.com (A100414)

• Antigen affinity-purified antibodies from Proteintech (19750-1-AP)

• Goat polyclonal antibodies from Santa Cruz Biotechnology (sc-131246)

Most of these antibodies are raised against synthetic peptides derived from human OR4K17, often targeting specific regions like the C-terminus or cytoplasmic domains. The majority are unconjugated, though secondary antibody options with various conjugates (HRP, FITC, biotin, etc.) are available for detection purposes .

What is the molecular weight of OR4K17 and how does this affect antibody selection?

The molecular weight of OR4K17 is approximately 34-35 kDa, based on both calculated and observed molecular weights in various experimental systems . When selecting an antibody, it's crucial to verify that the observed molecular weight in validation studies matches the expected size of OR4K17. This verification helps ensure specificity and reduces the risk of cross-reactivity with other olfactory receptors. Some antibodies show multiple bands due to post-translational modifications or alternative splicing, which may need to be considered when interpreting experimental results. Researchers should also verify that the antibody has been validated for their specific application and in relevant cell or tissue types that express OR4K17.

Which applications are OR4K17 antibodies validated for, and what are the recommended dilutions?

OR4K17 antibodies have been validated for multiple applications with specific recommended dilutions:

It's important to note that optimal dilutions may be sample-dependent and should be determined empirically for each experimental system. Antibodies have been validated in specific cell lines and tissues, including HepG2, HuH-7, L02 cells, human brain tissue for WB, and Jurkat cells for IF/ICC .

What are the optimal storage conditions for OR4K17 antibodies to maintain reactivity?

Most OR4K17 antibodies should be stored at -20°C for long-term preservation of reactivity . Upon receipt, it is recommended to aliquot the antibody to avoid repeated freeze-thaw cycles, which can degrade antibody performance. For example, the Proteintech antibody (19750-1-AP) is stable for one year after shipment when stored at -20°C, and aliquoting is unnecessary for this specific product .

The Antibodies.com product (A100414) is shipped at 4°C, but upon delivery should be aliquoted and stored at -20°C to avoid freeze/thaw cycles . Most OR4K17 antibodies are supplied in buffered solutions containing preservatives like sodium azide and stabilizers such as glycerol. The Proteintech antibody is provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 , while the Santa Cruz antibody contains PBS with <0.1% sodium azide and 0.1% gelatin .

How can I optimize immunofluorescence protocols when using OR4K17 antibodies?

Optimizing immunofluorescence protocols for OR4K17 requires attention to several factors:

• Fixation method: Different fixation protocols may affect epitope accessibility. Paraformaldehyde (4%) is commonly used, but methanol fixation might be preferred for membrane proteins like OR4K17.

• Permeabilization: Since OR4K17 is a multi-pass membrane protein, careful permeabilization is crucial. Use 0.1-0.3% Triton X-100 or 0.1% saponin depending on whether you're targeting intracellular or extracellular epitopes.

• Blocking: Thorough blocking (5% normal serum from the species of the secondary antibody) minimizes background, which is particularly important for olfactory receptors that may have homologs.

• Antibody dilution: Start with the manufacturer's recommended range (1:50-1:500) but optimize through titration. The Proteintech protocol suggests beginning at the higher concentration end (1:50) and adjusting as needed.

• Incubation times: Primary antibody incubation at 4°C overnight often yields better results than shorter incubations at room temperature.

• Controls: Include appropriate controls, such as a peptide competition assay. For example, Santa Cruz offers a blocking peptide (sc-131246 P) specifically for competition studies with their OR4K17 antibody .

• Secondary antibody selection: Choose appropriate conjugated secondary antibodies based on your detection system. For the Santa Cruz goat polyclonal, they recommend donkey anti-goat IgG-FITC (sc-2024) at 1:100-1:400 dilution or donkey anti-goat IgG-TR (sc-2783) at 1:100 dilution .

How can I confirm the specificity of OR4K17 antibody staining in my experiments?

Confirming OR4K17 antibody specificity requires multiple validation approaches:

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to your samples. This should abolish specific staining. Santa Cruz Biotechnology offers a blocking peptide (sc-131246 P) specifically for this purpose .

• Positive control tissues/cells: Use samples known to express OR4K17. Based on validation data, HepG2 cells, HuH-7 cells, L02 cells, and human brain tissue have been confirmed positive for Western blot, while Jurkat cells show positive staining in immunofluorescence .

• RNA interference: Perform siRNA knockdown experiments using OR4K17-specific siRNA (e.g., sc-92186) or shRNA (sc-92186-SH) to reduce target expression and confirm corresponding reduction in antibody signal .

• Molecular weight verification: Confirm that the detected band in Western blots appears at the expected molecular weight of approximately 35 kDa .

• Cross-reactivity assessment: Verify that the antibody doesn't cross-react with other OR4 family members. Some antibodies like the Santa Cruz sc-131246 are specifically noted to be non-cross-reactive with other OR4 family members .

• Multiple antibody comparison: Use different antibodies targeting distinct epitopes of OR4K17 to confirm staining patterns.

• Heterologous expression: Express tagged OR4K17 in a cell system and confirm co-localization of antibody staining with the tag.

What are the common challenges in detecting OR4K17 in different sample types?

Detection of OR4K17 presents several challenges that vary by sample type and experimental approach:

• Low endogenous expression levels: Olfactory receptors often show tissue-specific expression patterns and may be expressed at low levels in non-olfactory tissues, requiring sensitive detection methods and appropriate signal amplification.

• Membrane protein solubilization: As a multi-pass membrane protein, OR4K17 requires effective solubilization for Western blot analysis. Standard RIPA buffers may be insufficient; consider specialized membrane protein extraction protocols with appropriate detergents.

• Cross-reactivity with related receptors: The olfactory receptor family is the largest gene family in humans, with high sequence homology between members. This necessitates highly specific antibodies, such as those targeting unique epitopes or verified not to cross-react with other OR4 family members .

• Post-translational modifications: Like many GPCRs, OR4K17 may undergo various post-translational modifications that can affect antibody recognition or result in multiple bands/staining patterns.

• Fixation artifacts: In immunofluorescence applications, improper fixation can alter epitope accessibility or create artificial staining patterns. Optimization of fixation protocols is crucial, especially for membrane proteins.

• Background in immunofluorescence: When examining olfactory tissue, high autofluorescence may complicate analysis. Consider autofluorescence quenching protocols and careful selection of fluorophores with emission spectra distinct from tissue autofluorescence.

• Tissue-specific expression patterns: OR4K17 may show differential subcellular localization in different tissues, affecting the interpretation of staining patterns.

How can OR4K17 antibodies be used to study olfactory signal transduction pathways?

OR4K17 antibodies can be powerful tools for investigating olfactory signal transduction through multiple approaches:

• Co-immunoprecipitation (Co-IP): OR4K17 antibodies can be used to pull down the receptor and its associated protein complexes, helping identify binding partners in the signaling cascade. When coupled with mass spectrometry, this approach can reveal novel interactions between OR4K17 and other components of the olfactory signaling machinery.

• Proximity ligation assay (PLA): This technique can detect protein-protein interactions between OR4K17 and putative signaling partners with high specificity and sensitivity, providing spatial information about these interactions in intact cells.

• Dual immunofluorescence: Co-staining with OR4K17 antibodies and antibodies against components of the cAMP pathway (adenylate cyclase, PKA, CREB) can reveal co-localization and potential functional relationships. OR4K17 is known to signal through a cascade that leads to cAMP production via an olfactory-enriched adenylate cyclase .

• Calcium imaging combined with immunocytochemistry: Functional calcium imaging in response to odorants can be followed by OR4K17 immunostaining to correlate receptor expression with cellular responses.

• Phospho-specific antibody development: While not currently available commercially, antibodies specifically recognizing phosphorylated forms of OR4K17 could help track receptor activation status.

• BRET/FRET analyses: Using OR4K17 antibodies to validate expression of fluorescently tagged receptor constructs in resonance energy transfer experiments examining real-time protein interactions.

• Super-resolution microscopy: Advanced imaging techniques combined with highly specific OR4K17 antibodies can reveal the nanoscale organization of receptor clusters and their relationship to downstream signaling components.

What are the considerations for using OR4K17 antibodies in co-localization studies with other neuronal markers?

When designing co-localization studies involving OR4K17 and other neuronal markers, researchers should consider several important factors:

• Antibody species compatibility: Select primary antibodies raised in different host species to avoid cross-reactivity during secondary antibody detection. For example, if using the rabbit polyclonal OR4K17 antibody from Proteintech , pair it with mouse, goat, or rat-derived antibodies against other neuronal markers.

• Spectral overlap: Choose fluorophores with minimal spectral overlap for multi-color imaging. Consider the excitation/emission properties of each fluorophore and the filter sets available on your microscope.

• Sequential staining protocols: For challenging combinations, consider sequential rather than simultaneous staining, especially if antibodies require different fixation or antigen retrieval methods.

• Antibody penetration in tissue sections: OR4K17 is a membrane protein, so ensure sufficient permeabilization for antibody access while preserving tissue architecture.

• Controls for co-localization studies:

  • Single-labeled controls to assess bleed-through

  • Secondary antibody-only controls to assess non-specific binding

  • Absorption controls using blocking peptides where available

  • Quantitative co-localization analysis using appropriate software and statistical methods

• Subcellular localization considerations: OR4K17 is expected to localize primarily to the plasma membrane, particularly in the cilia of olfactory sensory neurons . When co-staining with markers for different subcellular compartments, consider the biological relevance of potential co-localization.

• Tissue autofluorescence: Olfactory tissue often exhibits significant autofluorescence. Implement appropriate quenching steps and include unstained tissue controls.

• Antibody sensitivity matching: Ensure that detection sensitivity is appropriately balanced between markers to avoid false negative co-localization due to differential sensitivity.

How might OR4K17 antibodies contribute to understanding ectopic expression of olfactory receptors?

Olfactory receptors, once thought to be exclusively expressed in nasal olfactory epithelium, are now known to be ectopically expressed in various non-olfactory tissues where they may serve novel functions. OR4K17 antibodies can play crucial roles in this emerging research area:

• Tissue expression profiling: Systematic immunohistochemical screening across human tissue arrays using validated OR4K17 antibodies can reveal unexpected expression patterns. Current data already shows OR4K17 expression in liver-derived cell lines (HepG2, HuH-7, L02) and brain tissue , suggesting functions beyond olfaction.

• Pathological significance assessment: Comparing OR4K17 expression in normal versus diseased tissues may reveal associations with specific pathologies. Antibodies with verified specificity are essential for such comparative studies.

• Functional characterization: Combined with functional assays and gene knockdown approaches, OR4K17 antibodies can help elucidate the receptor's role in non-olfactory tissues. The availability of siRNA and shRNA reagents specifically targeting OR4K17 facilitates such investigations .

• Single-cell analysis: Using OR4K17 antibodies for flow cytometry or single-cell immunohistochemistry can identify specific cell populations expressing this receptor within heterogeneous tissues, providing insights into cell-type specific functions.

• Developmental expression patterns: Studying OR4K17 expression across developmental stages may reveal temporal regulation patterns relevant to tissue development and differentiation.

• Subcellular localization in non-olfactory cells: While OR4K17 localizes to cilia in olfactory neurons , its subcellular distribution may differ in other cell types, potentially indicating alternative functions. High-resolution imaging with validated antibodies can reveal these patterns.

• Pathways and interactome analysis: Using OR4K17 antibodies for pull-down assays followed by proteomics can identify tissue-specific interaction partners, suggesting potential signaling pathways in non-olfactory contexts.

What are the technical considerations for developing multiplexed assays incorporating OR4K17 antibodies?

Developing multiplexed assays that include OR4K17 detection requires careful technical planning:

• Antibody compatibility in multiplex formats:

  • Ensure compatible fixation requirements across all target proteins

  • Verify that denaturing conditions for one target don't disrupt epitopes for others

  • Consider antibody cross-reactivity, particularly when using multiple rabbit polyclonal antibodies

• Signal amplification and normalization:

  • For low-abundance targets like OR4K17, incorporate appropriate signal amplification methods (tyramide signal amplification, polymer detection systems)

  • Include internal controls for normalization across different experimental conditions

• Platform-specific considerations:

  • For protein array approaches: Validate OR4K17 antibody specificity in array formats, which may differ from traditional Western blot or IF applications

  • For multiplex flow cytometry: Optimize permeabilization protocols for this membrane protein while preserving other epitopes

  • For multiplex immunohistochemistry: Consider sequential antibody application and stripping protocols

• Quantification methods:

  • Establish appropriate quantification metrics for OR4K17 in the context of other measured proteins

  • Develop standard curves using recombinant OR4K17 protein where available

  • Account for potential non-linear relationships in signal intensity

• Validation strategies for multiplexed data:

  • Confirm key findings using orthogonal single-plex methods

  • Include biological controls with known expression patterns

  • Consider spike-in controls with known quantities of target proteins

• Data analysis approaches:

  • Implement appropriate statistical methods for high-dimensional data

  • Consider machine learning approaches for pattern recognition in complex datasets

  • Develop visualization tools that effectively communicate relationships between OR4K17 and other measured proteins

• Emerging multiplexed technologies:

  • Explore compatibility with cutting-edge approaches like Imaging Mass Cytometry, CODEX, or Digital Spatial Profiling

  • Consider development of OR4K17-specific aptamers as alternative affinity reagents for multiplexed applications