ORTH2 Antibody

What is OR2H2 and why is it a target for antibody development?

OR2H2 (Olfactory Receptor Family 2 Subfamily H Member 2) is an odorant receptor protein with a calculated molecular weight of 34,763 Da. This G-protein coupled receptor belongs to the G-protein coupled receptor 1 family. The scientific interest in OR2H2 stems from its role in olfactory signaling pathways and potential involvement in non-olfactory tissues, making it a valuable research target for understanding sensory perception mechanisms and potentially unexplored physiological functions. Antibodies targeting OR2H2 allow researchers to detect, quantify, and visualize this protein in various experimental contexts .

What are the key characteristics of the OR2H2 antibody?

The commercially available anti-OR2H2 antibody (C-term) is typically a polyclonal antibody generated in rabbits using a KLH-conjugated synthetic peptide derived from the C-terminal region (amino acids 276-302) of human OR2H2. It is provided as a purified polyclonal antibody in PBS with 0.09% (W/V) sodium azide. This antibody has demonstrated reactivity specifically with human OR2H2 and has been validated for Western Blot (WB) and Flow Cytometry (FC) applications .

How should the OR2H2 antibody be stored to maintain its activity?

For optimal preservation of antibody activity, anti-OR2H2 antibody should be maintained at 2-8°C for short-term storage (up to 2 weeks). For long-term storage, the recommended approach is to divide the antibody into small aliquots and store at -20°C to prevent repeated freeze-thaw cycles that can degrade antibody quality and performance. This storage protocol helps maintain the structural integrity and binding capacity of the antibody, ensuring consistent experimental results over time .

What are the validated applications for OR2H2 antibody in research?

The OR2H2 antibody has been rigorously validated for Western Blotting (WB) and Flow Cytometry (FC) applications. In Western Blotting, the antibody has successfully detected OR2H2 protein in multiple human cell lines including HepG2, K562, 293, MCF-7, and LNCap, as well as in human skeletal muscle tissue. For Flow Cytometry, the antibody has been verified using HepG2 cells. While these are the validated applications, researchers may conduct pilot studies to adapt the antibody for other potential applications such as immunohistochemistry (IHC), immunocytochemistry (ICC), or immunoprecipitation (IP) .

What are the recommended dilutions and protocols for Western Blotting with OR2H2 antibody?

For Western Blotting applications, the recommended dilution for the OR2H2 antibody is 1:2000. A validated protocol involves:

• Sample preparation: Prepare whole cell lysates from target cells (20 μg protein per lane)

• SDS-PAGE: Separate proteins using standard gel electrophoresis

• Transfer: Transfer proteins to a membrane using standard methods

• Blocking: Block membrane with 5% non-fat dry milk in TBST

• Primary antibody: Incubate with anti-OR2H2 antibody at 1:2000 dilution

• Secondary antibody: Use goat anti-rabbit IgG (H+L) conjugated to peroxidase at 1:10000 dilution

• Detection: Visualize using appropriate chemiluminescent detection system

The expected band size for OR2H2 protein is approximately 35 kDa. This protocol has been validated with multiple cell lines including HepG2, K562, 293, MCF-7, and LNCap .

What methodology should be followed for Flow Cytometry using OR2H2 antibody?

For Flow Cytometry applications, researchers should use a dilution range of 1:10 to 1:50 for the OR2H2 antibody. The validated methodology includes:

• Cell preparation: Harvest and wash cells in cold PBS

• Fixation/permeabilization: Fix and permeabilize cells using appropriate reagents

• Blocking: Block with normal serum to reduce non-specific binding

• Primary antibody: Incubate cells with anti-OR2H2 antibody (1:10-1:50 dilution)

• Wash: Remove unbound primary antibody

• Secondary antibody: Incubate with FITC-conjugated goat-anti-rabbit secondary antibody

• Final wash: Remove unbound secondary antibody

• Analysis: Analyze using flow cytometer with appropriate controls

This protocol has been successfully applied to HepG2 cells, demonstrating specific detection of OR2H2 protein .

How should positive and negative controls be selected for OR2H2 antibody experiments?

Selecting appropriate controls is crucial for validating experimental results with OR2H2 antibody:

Positive Controls:

• HepG2, K562, 293, MCF-7, and LNCap cell lines have been validated to express OR2H2 and can serve as positive controls

• Human skeletal muscle tissue has also shown OR2H2 expression and can be used as a tissue-based positive control

Negative Controls:

• Cell lines without OR2H2 expression (researchers should verify through literature or preliminary testing)

• Primary antibody omission controls to assess non-specific binding of secondary antibody

• Isotype controls using non-specific rabbit IgG at the same concentration as the OR2H2 antibody

• Peptide competition assays using the immunizing peptide to confirm specificity

Including these controls allows researchers to accurately assess antibody specificity and validate experimental findings .

What factors should be considered when designing cross-reactivity studies for OR2H2 antibody?

When assessing potential cross-reactivity of OR2H2 antibody, researchers should consider:

• Sequence homology analysis: Compare the immunizing peptide sequence (amino acids 276-302 of human OR2H2) with other olfactory receptors and G-protein coupled receptors to identify potential cross-reactive proteins

• Species cross-reactivity: While the antibody is validated for human OR2H2, researchers working with other species should perform preliminary validation studies

• Alternative splicing: Consider potential splice variants of OR2H2 that may or may not contain the C-terminal epitope

• Cell lines with varying OR2H2 expression levels: Include cell lines with high, moderate, low, and no expression of OR2H2

• Western blot analysis with blocking peptide: Perform parallel Western blots with and without pre-incubation with the immunizing peptide to confirm band specificity

Thorough cross-reactivity testing enhances confidence in experimental findings and prevents misinterpretation of results .

How can researchers optimize immunostaining protocols for detecting OR2H2 in different tissue types?

While the OR2H2 antibody has not been specifically validated for immunohistochemistry, researchers can adapt existing protocols with these optimization considerations:

• Fixation method: Compare different fixatives (formalin, paraformaldehyde, methanol) to determine optimal preservation of the OR2H2 epitope

• Antigen retrieval: Test various antigen retrieval methods (heat-induced in citrate buffer, EDTA buffer, or enzymatic digestion)

• Blocking conditions: Optimize blocking solutions to minimize background (BSA, normal serum, commercial blockers)

• Antibody concentration: Perform a titration experiment with dilutions ranging from 1:100 to 1:1000

• Incubation conditions: Compare different temperatures (4°C, room temperature, 37°C) and durations (1 hour, overnight)

• Detection system: Evaluate different visualization methods (fluorescent vs. enzymatic)

• Counterstains: Select appropriate nuclear and cytoplasmic counterstains that don't interfere with OR2H2 signal

Methodical optimization of these parameters will yield protocols tailored to specific tissue types and research questions .

How can researchers validate the specificity of bands detected in Western blots using OR2H2 antibody?

To validate band specificity in Western blots:

• Molecular weight verification: Confirm that the detected band corresponds to the expected molecular weight of OR2H2 (approximately 35 kDa)

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide; specific bands should disappear or be significantly reduced

• siRNA knockdown: Perform parallel Western blots on control cells and cells with OR2H2 knocked down via siRNA; specific bands should show reduced intensity in knockdown samples

• Multiple antibody approach: Use a second antibody targeting a different epitope of OR2H2 to confirm detection of the same protein

• Mass spectrometry: Excise the band of interest and perform mass spectrometry analysis to confirm protein identity

This multi-faceted approach provides robust validation of band specificity and enhances confidence in experimental results .

What are common sources of background in OR2H2 antibody experiments and how can they be addressed?

Common sources of background and their solutions include:

Systematic troubleshooting of these issues can significantly improve signal-to-noise ratio in experiments utilizing OR2H2 antibody .

How should researchers interpret discrepancies between OR2H2 protein expression levels detected by different methods?

When faced with discrepancies between methods (e.g., Western blot vs. Flow Cytometry):

• Consider epitope accessibility: Different experimental conditions may affect exposure of the C-terminal epitope recognized by the antibody

• Evaluate protein conformation: Native vs. denatured states may affect antibody binding efficiency

• Assess method sensitivity: Flow cytometry may detect lower expression levels than Western blotting

• Post-translational modifications: These may affect antibody binding in certain experimental contexts

• Sample preparation differences: Cell lysis methods for Western blot vs. fixation for flow cytometry can impact epitope preservation

• Quantification approach: Compare the quantification methods used for each technique

Researchers should triangulate results using multiple methods and consider biological context when interpreting discrepancies, rather than relying on a single approach .

How can OR2H2 antibody be utilized in co-immunoprecipitation studies to identify protein-protein interactions?

While not specifically validated for immunoprecipitation, researchers can adapt the OR2H2 antibody for co-IP studies with the following methodology:

• Optimization of lysis conditions: Use non-denaturing buffers that preserve protein-protein interactions while effectively extracting membrane-bound OR2H2

• Antibody immobilization: Conjugate OR2H2 antibody to protein A/G beads or magnetic beads

• Pre-clearing lysates: Remove non-specific binding proteins by pre-incubating lysates with beads alone

• Immunoprecipitation: Incubate cleared lysates with antibody-conjugated beads

• Washing stringency optimization: Balance between removing non-specific interactions and preserving true interactions

• Elution methods: Compare different elution strategies (low pH, competitive elution with peptide, boiling in sample buffer)

• Detection methods: Use sensitive detection methods for Western blot analysis of co-precipitated proteins

• Validation: Confirm interactions using reverse co-IP and additional methods like proximity ligation assay

This approach can reveal novel interaction partners of OR2H2, potentially uncovering new functions beyond its known olfactory role .

What considerations should be taken into account when using OR2H2 antibody for studying post-translational modifications?

When investigating post-translational modifications (PTMs) of OR2H2:

• Epitope location: Consider whether the C-terminal epitope (aa 276-302) contains or is adjacent to known/predicted PTM sites

• Phosphorylation studies: Use phosphatase inhibitors during sample preparation; consider phospho-specific antibodies as complementary tools

• Glycosylation analysis: Compare molecular weights before and after deglycosylation treatment

• Ubiquitination detection: Use proteasome inhibitors to enhance detection of ubiquitinated forms

• PTM-preserving lysis buffers: Select buffers containing appropriate inhibitors for the PTM of interest

• Resolution optimization: Use high-resolution gel systems or specialized gradient gels to separate modified forms

• Mass spectrometry validation: Confirm PTM sites through immunoprecipitation followed by mass spectrometry

• Functional correlation: Design experiments to correlate detected PTMs with functional outcomes

Understanding OR2H2 PTMs may provide insights into receptor regulation, trafficking, and signaling mechanisms .

How can OR2H2 antibody be integrated into advanced imaging techniques for studying receptor localization and trafficking?

For advanced imaging applications with OR2H2 antibody:

• Super-resolution microscopy optimization:

  • Sample preparation: Test different fixation and permeabilization protocols to preserve membrane structures

  • Signal enhancement: Explore signal amplification methods for optimal detection

  • Multicolor imaging: Combine OR2H2 antibody with markers for cellular compartments

• Live-cell imaging adaptations:

  • Antibody fragment generation: Consider creating Fab fragments for improved penetration

  • Fluorophore selection: Use photostable fluorophores with appropriate spectral properties

  • Internalization studies: Design pulse-chase experiments to track receptor trafficking

• Colocalization studies:

  • Marker selection: Use established markers for endoplasmic reticulum, Golgi, plasma membrane, and endocytic compartments

  • Quantification methods: Apply rigorous colocalization analysis (Pearson's coefficient, Manders' overlap coefficient)

  • 3D reconstruction: Collect z-stacks for complete spatial analysis of receptor distribution

• Proximity-based techniques:

  • FRET microscopy: Combine OR2H2 antibody with antibodies against potential interaction partners

  • Proximity ligation assay: Detect and quantify protein interactions with spatial resolution

These advanced imaging approaches can reveal dynamic aspects of OR2H2 biology including trafficking, internalization, and compartmentalization .

How might OR2H2 antibody be utilized in studying the functional diversity of olfactory receptors across different tissues?

Recent research suggests olfactory receptors may have functions beyond their canonical role in olfaction. For studying OR2H2 across tissues:

• Tissue expression profiling:

  • Use OR2H2 antibody for Western blot analysis across tissue panels

  • Develop tissue microarray immunostaining protocols

  • Correlate protein expression with existing transcriptomic data

• Functional studies in non-olfactory tissues:

  • Combine OR2H2 detection with functional readouts (calcium imaging, cAMP assays)

  • Design co-immunoprecipitation studies to identify tissue-specific interaction partners

  • Develop cell-specific knockout models followed by antibody validation

• Comparative analysis:

  • Study OR2H2 expression patterns in health vs. disease states

  • Investigate developmental changes in expression and localization

  • Compare subcellular localization across different cell types

This research direction could reveal novel physiological functions of OR2H2 beyond olfactory sensing, potentially identifying new therapeutic targets .

What methodological approaches can integrate OR2H2 antibody with gene editing technologies for receptor characterization?

Combining OR2H2 antibody with gene editing technologies offers powerful research capabilities:

• CRISPR/Cas9 epitope tagging:

  • Generate epitope-tagged OR2H2 (FLAG, HA, GFP) for complementary detection approaches

  • Validate tagged constructs against antibody-based detection

  • Develop dual-detection systems for enhanced specificity

• Knockout validation:

  • Create OR2H2 knockout cell lines as definitive negative controls

  • Use antibody to confirm complete protein loss

  • Perform rescue experiments with wild-type and mutant constructs

• Domain-specific mutagenesis:

  • Generate partial deletions or point mutations in the C-terminal region

  • Use OR2H2 antibody to assess effects on expression, localization, and stability

  • Correlate structural modifications with functional outcomes

• Single-cell analysis:

  • Combine gene editing with antibody-based detection in single-cell experimental designs

  • Correlate genotype with protein expression at single-cell resolution

  • Develop heterogeneous expression systems for comparative analysis

These integrated approaches enable precise characterization of OR2H2 structure-function relationships and regulatory mechanisms .

How can researchers apply computational approaches to enhance OR2H2 antibody experimental design and interpretation?

Computational methods can significantly enhance OR2H2 antibody research:

• Epitope prediction and analysis:

  • Analyze the C-terminal peptide (aa 276-302) for structural properties and accessibility

  • Predict potential post-translational modification sites within the epitope region

  • Model the antibody-epitope interaction to understand binding determinants

• Cross-reactivity assessment:

  • Perform comprehensive sequence alignment with related olfactory receptors

  • Identify potential cross-reactive epitopes through similarity scoring

  • Design experiments to specifically test predicted cross-reactions

• Systems biology integration:

  • Incorporate antibody-derived protein expression data into pathway analyses

  • Correlate OR2H2 expression with transcriptomic and proteomic datasets

  • Model potential signaling networks based on experimental findings

• Machine learning applications:

  • Develop image analysis algorithms for automated quantification of immunostaining

  • Train models to recognize subcellular localization patterns

  • Create predictive models of OR2H2 function based on expression patterns

These computational approaches enhance experimental rigor and facilitate the integration of OR2H2 research into broader biological contexts .

What strategies can researchers employ when facing inconsistent OR2H2 antibody performance across different batches?

Batch-to-batch variability is a common challenge with polyclonal antibodies. Researchers can address this through:

• Reference sample validation:

  • Maintain a stock of positive control lysate (e.g., HepG2 cells) to test each new antibody batch

  • Document band intensity and pattern for comparative evaluation

  • Establish acceptance criteria for batch qualification

• Standardization approaches:

  • Normalize antibody concentration before use (protein assay)

  • Perform titration experiments with each new batch

  • Consider pooling small aliquots from different batches for long-term projects

• Protocol adaptation:

  • Adjust incubation time and temperature for optimal performance

  • Modify blocking conditions to address batch-specific background

  • Fine-tune detection systems based on signal strength

• Alternative validation:

  • Confirm key findings with alternative detection methods

  • Consider monoclonal alternatives if available for critical experiments

  • Use peptide competition assays to confirm specificity of each batch

These approaches ensure experimental continuity despite inherent variability in polyclonal antibody production .

How should researchers interpret and address potential discrepancies between mRNA and protein expression levels of OR2H2?

Discrepancies between OR2H2 mRNA and protein levels detected by antibody-based methods may arise from several factors:

• Post-transcriptional regulation:

  • Investigate microRNA-mediated regulation of OR2H2 mRNA

  • Assess mRNA stability through actinomycin D chase experiments

  • Examine translation efficiency using polysome profiling

• Protein turnover dynamics:

  • Measure protein half-life using cycloheximide chase assays

  • Investigate proteasomal and lysosomal degradation pathways

  • Assess the impact of cellular stress on OR2H2 stability

• Technical considerations:

  • Compare sensitivity limits of RT-qPCR vs. antibody-based detection

  • Evaluate potential impact of sample preparation on protein preservation

  • Consider timing of analysis (mRNA changes may precede protein changes)

• Methodological approach:

  • Perform time-course experiments to capture dynamic relationships

  • Use single-cell approaches to address population heterogeneity

  • Develop quantitative standards for both mRNA and protein measurements

Understanding these discrepancies can reveal important regulatory mechanisms controlling OR2H2 expression and function .

What approaches can be used to optimize OR2H2 antibody performance in challenging samples like primary tissues or patient-derived specimens?

Working with OR2H2 antibody in complex samples requires specialized approaches:

• Sample preservation optimization:

  • Compare different fixation methods (fresh-frozen vs. FFPE)

  • Evaluate preservation solutions that maintain membrane protein integrity

  • Develop rapid processing protocols to minimize protein degradation

• Extraction method refinement:

  • Test specialized membrane protein extraction buffers

  • Optimize detergent type and concentration for efficient solubilization

  • Consider sequential extraction to enrich OR2H2-containing fractions

• Signal enhancement strategies:

  • Implement tyramide signal amplification for low abundance detection

  • Use polymer-based detection systems for improved sensitivity

  • Consider automated staining platforms for consistency

• Background reduction techniques:

  • Develop tissue-specific blocking protocols (including endogenous biotin blocking)

  • Preabsorb antibody with tissue homogenates to remove non-specific binding

  • Use antigen retrieval optimization matrices to determine optimal conditions

• Validation approaches:

  • Compare multiple antibody dilutions and incubation conditions

  • Include known positive and negative tissue controls

  • Correlate with in situ hybridization or RNAscope for mRNA detection

These optimizations enable reliable OR2H2 detection in challenging samples, expanding research possibilities beyond cell line models .

How might advances in antibody engineering enhance next-generation OR2H2 detection tools?

Emerging antibody technologies offer promising improvements for OR2H2 research:

• Recombinant antibody development:

  • Generation of single-chain variable fragments (scFvs) against OR2H2

  • Creation of bispecific antibodies targeting OR2H2 and subcellular markers

  • Development of camelid nanobodies for improved penetration in intact tissues

• Site-specific conjugation:

  • Engineered antibodies with controlled fluorophore attachment sites

  • Optimized antibody-drug conjugates for functional studies

  • Click chemistry-compatible antibodies for modular labeling

• Affinity maturation:

  • In vitro evolution to generate higher-affinity OR2H2 binders

  • Development of conformation-specific antibodies

  • Creation of antibodies with reduced cross-reactivity to related receptors

• Multimodal capabilities:

  • Dual-function antibodies for simultaneous detection and pull-down

  • Photoactivatable antibodies for spatiotemporal control of binding

  • Antibody-based biosensors for live monitoring of OR2H2 dynamics

These advances will expand the research toolkit beyond current polyclonal antibodies, enabling more sophisticated investigations of OR2H2 biology .

What emerging applications might benefit from integrating OR2H2 antibody research with single-cell technologies?

The intersection of OR2H2 antibody research with single-cell approaches offers exciting possibilities:

• Single-cell proteomics:

  • Antibody-based mass cytometry (CyTOF) including OR2H2 detection

  • Integration with single-cell transcriptomics for multi-omic analysis

  • Development of antibody panels to characterize OR2H2-expressing cell populations

• Spatial transcriptomics integration:

  • Combine OR2H2 antibody staining with spatial transcriptomics

  • Map receptor distribution in complex tissues with spatial context

  • Correlate protein localization with local transcriptional environments

• Microfluidic applications:

  • Single-cell Western blotting for quantitative OR2H2 protein analysis

  • Droplet-based antibody assays for high-throughput screening

  • Microfluidic tissue culture with integrated antibody-based detection

• Lineage tracing:

  • Use OR2H2 antibody to identify and isolate specific cell populations

  • Track developmental trajectories of OR2H2-expressing cells

  • Study clonal evolution in cancer models expressing OR2H2

These integrative approaches will provide unprecedented resolution in understanding the heterogeneity of OR2H2 expression and function across diverse cell populations .

How can OR2H2 antibody research contribute to understanding the broader olfactory receptor family functioning beyond sensory perception?

Expanding OR2H2 research beyond traditional boundaries can advance understanding of the entire olfactory receptor family:

• Comparative receptor studies:

  • Develop standardized antibody-based methods applicable across receptor family members

  • Create expression atlases spanning multiple olfactory receptors

  • Identify common regulatory mechanisms and trafficking pathways

• Non-canonical signaling investigation:

  • Use OR2H2 as a model to explore G-protein independent signaling

  • Study cross-talk between olfactory receptors and other signaling pathways

  • Investigate potential roles in developmental processes

• Physiological function exploration:

  • Examine OR2H2 and related receptors in metabolic regulation

  • Investigate potential roles in immune function

  • Study possible contributions to neurological processes

• Therapeutic target assessment:

  • Evaluate OR2H2 as a representative member for drug discovery approaches

  • Develop screening platforms using antibody-based detection

  • Create receptor-specific modulators based on structural insights

This broader perspective will position OR2H2 research within a systems biology framework, potentially revealing unexpected functions of olfactory receptors throughout the body .