OR4K2 Antibody

What is OR4K2 and why is it studied in research?

OR4K2 (Olfactory Receptor Family 4 Subfamily K Member 2) is a G-protein coupled receptor involved in olfactory sensing. It belongs to the large family of olfactory receptors that interact with odorant molecules in the nose to initiate neuronal responses that trigger smell perception. OR4K2 is a multi-pass membrane protein with a molecular weight of approximately 35kDa .

Research on OR4K2 is valuable for understanding:

• The mechanisms of olfactory signal transduction

• Structure and function of G-protein coupled receptors

• The genetics and evolution of the olfactory receptor gene family, which is the largest in the genome

• Potential roles in non-olfactory tissues, as ectopic expression has been documented

How should I select the appropriate OR4K2 antibody for my specific experimental application?

Selection should be based on several critical factors:

• Application compatibility: Different antibodies perform optimally in different applications:

  • For Western blot: Look for antibodies validated specifically for WB with recommended dilutions of 1:500-1:2000

  • For immunofluorescence: Choose antibodies validated for IF with dilutions typically around 1:200-1:1000

  • For ELISA applications: Select antibodies tested for ELISA with appropriate dilutions (often much higher, e.g., 1:40000)

• Species cross-reactivity: Ensure the antibody recognizes OR4K2 in your experimental organism:

  • Many OR4K2 antibodies are human-specific

  • Some antibodies cross-react with mouse and rat OR4K2

• Epitope considerations: Most OR4K2 antibodies target the C-terminal region. This is important when:

  • Creating fusion proteins (avoid C-terminal tags if using these antibodies)

  • Studying truncated variants

  • Investigating post-translational modifications

• Validation data: Prioritize antibodies with substantial validation:

  • Western blot validation showing the expected 35kDa band

  • Negative controls with blocking peptides

  • Cross-validation with different antibodies or techniques

What are the recommended protocols and conditions for Western blot detection of OR4K2?

Based on published methodologies:

• Sample preparation:

  • Cell lysates from Jurkat or HT-29 cells have been successfully used

  • Use standard lysis buffers containing protease inhibitors

  • For membrane proteins like OR4K2, ensure complete solubilization with appropriate detergents

• Protein denaturation and loading:

  • Heat samples at 95°C for 5 minutes in Laemmli buffer

  • Load 20-40μg total protein per lane

• Gel electrophoresis and transfer:

  • 10-12% SDS-PAGE gels are suitable for resolving the 35kDa OR4K2 protein

  • Transfer to PVDF membranes at 100V for 1 hour or 30V overnight

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with primary OR4K2 antibody at 1:500-1:2000 dilution

  • Incubate overnight at 4°C or 2 hours at room temperature

  • After washing, use appropriate HRP-conjugated secondary antibodies

• Detection controls:

  • Use blocking peptide as negative control to confirm specificity

  • Consider known OR4K2-expressing cell lines as positive controls

The expected result is a band at approximately 35kDa, which corresponds to the full-length OR4K2 protein.

What are common challenges in OR4K2 protein detection and how can they be addressed?

Common Challenge 1: Weak or no signal in Western blot

Potential solutions:

• Increase antibody concentration (use 1:500 instead of 1:2000)

• Extend primary antibody incubation time to overnight at 4°C

• Verify protein expression levels in your experimental system (OR4K2 may have tissue-specific expression)

• Use fresh antibody aliquots and avoid repeated freeze-thaw cycles

• Consider using enhanced chemiluminescence (ECL) substrates with higher sensitivity

• Optimize protein extraction protocol for membrane proteins using appropriate detergents

Common Challenge 2: Multiple bands or high background

Potential solutions:

• Increase blocking time or concentration (try 5% BSA instead of milk)

• Extend wash steps (5 x 5 minutes with TBST)

• Decrease primary antibody concentration (try 1:2000 instead of 1:500)

• Use fresh blocking solutions

• Pre-absorb antibody with non-specific proteins

• Consider using monoclonal antibodies for higher specificity

Common Challenge 3: Inconsistent immunostaining results

Potential solutions:

• Standardize fixation protocols (4% paraformaldehyde for 15 minutes is commonly effective)

• Optimize permeabilization conditions for membrane proteins

• Use positive control samples with known OR4K2 expression

• Consider antigen retrieval methods if working with fixed tissues

How should OR4K2 antibodies be stored and handled to maintain optimal activity?

Based on manufacturer recommendations :

• Storage conditions:

  • Store at -20°C for long-term storage (maintains activity for approximately 1 year)

  • Avoid repeated freeze-thaw cycles

  • Consider preparing single-use aliquots upon receipt

• Shipping and handling:

  • Antibodies are typically shipped at 4°C

  • Upon receipt, store according to manufacturer's instructions

  • Some antibodies are supplied in stabilizing solutions containing 50% glycerol, which helps prevent freeze-thaw damage

• Working solution preparation:

  • Dilute in fresh buffer solutions immediately before use

  • For ELISA applications, prepare dilutions in appropriate assay buffers

  • When working with diluted antibody solutions, maintain cold temperatures and use within 24 hours

How can OR4K2 antibodies be used in single-cell analysis and subcellular localization studies?

Single-cell and subcellular localization studies require specialized approaches:

• Immunofluorescence optimization for subcellular localization:

  • Use confocal microscopy for precise localization

  • Co-stain with organelle markers (e.g., membrane markers like Na+/K+ ATPase)

  • Dilute OR4K2 antibodies at 1:200-1:1000 for optimal signal-to-noise ratio

  • Consider super-resolution microscopy techniques for detailed membrane localization

  • The Human Protein Atlas provides subcellular localization data that can serve as reference

• Single-cell analysis techniques:

  • Flow cytometry can be used with permeabilized cells and appropriate fluorophore-conjugated secondary antibodies

  • For single-cell transcriptomics correlation, combine with RNA-FISH techniques

  • Mass cytometry (CyTOF) may be employed for multiple parameter analysis at single-cell resolution

  • Single-cell proteomic approaches can validate antibody binding at individual cell level

• Considerations for olfactory receptor trafficking:

  • OR4K2, like other olfactory receptors, undergoes complex trafficking

  • Use pulse-chase experiments with OR4K2 antibodies to track protein movement

  • Live-cell imaging may require creation of tagged OR4K2 constructs for validation

How can OR4K2 antibodies be validated for cross-reactivity and specificity?

Comprehensive validation approaches include:

• Genetic validation methods:

  • Use CRISPR/Cas9 knockout cell lines lacking OR4K2 as negative controls

  • siRNA knockdown experiments to demonstrate specificity

  • Overexpression systems with tagged OR4K2 for co-localization studies

• Biochemical validation:

  • Pre-absorption with immunizing peptide should eliminate specific signal

  • Mass spectrometry validation of immunoprecipitated proteins

  • Epitope mapping to confirm binding to expected protein regions

  • Competition assays with known binders

• Cross-reactivity assessment:

  • Test antibodies on tissues/cells from different species if cross-reactivity is claimed

  • Evaluate potential cross-reactivity with closely related olfactory receptors

  • Perform sequence alignment analysis of the immunogen peptide against related proteins

• Technical validation:

  • Compare results from multiple antibodies targeting different epitopes

  • Use different detection methods (e.g., WB, IF, ELISA) to confirm consistency

  • Validate with orthogonal methods like RNA expression data

Can OR4K2 antibodies be used in studies of olfactory dysfunction in disease models?

Several methodological approaches can be employed:

• Analysis of receptor expression in disease models:

  • Quantitative Western blot analysis using calibrated OR4K2 antibodies

  • Immunohistochemistry of olfactory epithelium in normal vs. diseased tissue

  • Flow cytometry for quantifying receptor-positive cells in disaggregated tissues

• Functional correlation studies:

  • Combine antibody labeling with calcium imaging or electrophysiology

  • Correlate receptor expression levels with olfactory sensory neuron activity

  • Use in animal models of olfactory dysfunction (e.g., aging, neurodegenerative diseases)

• Potential limitations and controls:

  • Species differences in OR4K2 sequence may limit use in some animal models

  • Include age-matched controls when studying developmental or aging effects

  • Consider the natural variability in olfactory receptor expression

How can OR4K2 antibodies be incorporated into high-throughput or multiplexed detection systems?

Modern multiplexed approaches include:

• Antibody array systems:

  • OR4K2 antibodies can be spotted onto antibody microarrays

  • Include appropriate controls and standardization curves

  • Validate signal specificity in multiplexed format separately

• Multiplexed immunofluorescence techniques:

  • Sequential immunostaining with OR4K2 antibodies and other markers

  • Spectral unmixing for multiple fluorophores

  • Cyclic immunofluorescence for highly multiplexed imaging

• Mass cytometry and imaging mass cytometry:

  • Metal-conjugated OR4K2 antibodies can be used in CyTOF systems

  • Enables simultaneous detection of dozens of proteins

  • Requires careful titration and validation in the multiplexed context

• ELISA-based multiplexing:

  • Incorporation into bead-based multiplexed ELISA platforms

  • Use with appropriate dilutions (typically 1:40000)

  • Test for cross-reactivity with other antibodies in the multiplex panel

How should researchers interpret quantitative data from OR4K2 antibody-based experiments?

Guidelines for rigorous data interpretation:

• Western blot quantification approaches:

  • Use total protein normalization rather than single housekeeping genes

  • Employ linear range detection methods

  • Include calibration curves with recombinant standards when possible

  • Present data as fold-change relative to appropriate controls

• ELISA data analysis:

  • Working range for OR4K2 ELISA kits is typically 0.156-10 ng/ml

  • Ensure samples fall within the linear range of the standard curve

  • Consider the effect of sample matrix on antibody binding

  • Account for potential interfering substances in complex biological samples

• Statistical considerations:

  • Perform appropriate statistical tests based on data distribution

  • Consider biological vs. technical replicates in experimental design

  • Use power analysis to determine adequate sample sizes

  • Account for batch effects in multi-experiment analyses

How can results from OR4K2 antibody experiments be integrated with other molecular data?

Integrative approaches include:

• Multi-omics integration methods:

  • Correlate protein expression data from antibody-based detection with transcriptomic data

  • Integrate with genomic data on OR4K2 genetic variants

  • Combine with metabolomic data for functional pathway analysis

  • Use machine learning approaches for pattern recognition across multiple data types

• Pathway and network analysis:

  • Place OR4K2 protein data in context of G-protein coupled receptor signaling networks

  • Consider interaction partners identified through co-immunoprecipitation

  • Map to known olfactory transduction pathways

  • Use pathway enrichment analysis for functional interpretation

• Visualization techniques:

  • Create multi-dimensional visualizations incorporating OR4K2 antibody data

  • Use heat maps for comparing expression across different conditions

  • Develop interactive visualizations for exploring relationships between variables

  • Consider temporal aspects when integrating time-course data

How might advances in antibody engineering impact future OR4K2 research?

Emerging antibody technologies with potential applications include:

• Single-domain antibodies and nanobodies:

  • Smaller size allows better penetration into tissues and access to cryptic epitopes

  • Potential for detecting conformational states of OR4K2 as a GPCR

  • May provide new tools for studying OR4K2 in live-cell applications

• Recombinant antibody technologies:

  • Phage display for generating highly specific OR4K2 antibodies

  • Recent advances in genotype-phenotype linked antibody screening systems can accelerate discovery

  • Synthetic antibody libraries may overcome immunogenicity limitations

• Engineered antibody fragments:

  • Fab and scFv fragments for improved tissue penetration

  • Bispecific antibodies for co-detection of OR4K2 with signaling partners

  • Intrabodies for tracking OR4K2 in living cells

What are emerging applications of OR4K2 antibodies beyond traditional olfactory research?

Novel research directions include:

• Ectopic expression studies:

  • Investigation of OR4K2 expression in non-olfactory tissues

  • Potential roles in sperm chemotaxis or other chemosensory functions

  • Connections to other GPCR signaling networks in diverse cell types

• Drug discovery applications:

  • Antibody-based screening for compounds that modulate OR4K2 activity

  • Conformational antibodies that distinguish active vs. inactive receptor states

  • Target validation for olfactory-based therapeutics

• Methodological innovations:

  • Proximity labeling approaches using OR4K2 antibodies

  • CRISPR-based tagging for endogenous tracking

  • Optogenetic or chemogenetic tools combined with antibody validation