OR2T29/OR2T5 Antibody

What are OR2T29 and OR2T5 proteins, and why are antibodies against them significant in research?

OR2T29 and OR2T5 are olfactory receptors belonging to the G-protein coupled receptor (GPCR) family. These receptors interact with odorant molecules in the nose to initiate neuronal responses that trigger smell perception. They share a 7-transmembrane domain structure with many neurotransmitter and hormone receptors and are responsible for the recognition and G protein-mediated transduction of odorant signals .

The olfactory receptor gene family is the largest in the genome, with OR2T29 and OR2T5 being coded by single-exon genes. Antibodies against these receptors serve as essential tools for studying their expression patterns in different tissues, investigating olfactory perception mechanisms, exploring potential involvement in neurological conditions, and examining receptor trafficking and signaling pathways.

Recent research has shown an enrichment of olfactory receptor genes in genome-wide studies of autism spectrum disorders, highlighting the potential importance of these receptors beyond canonical roles in olfaction .

What types of OR2T29/OR2T5 antibodies are currently available for research applications?

Several types of OR2T29/OR2T5 antibodies are available for research, each with specific characteristics:

The choice between monoclonal and polyclonal antibodies depends on research requirements:

Most commercially available antibodies target synthetic peptides derived from specific regions of the human OR2T5/OR2T29 proteins, particularly the amino acid regions 56-115 .

How should OR2T29/OR2T5 antibodies be optimized for Western Blot experiments?

Western blot analysis using OR2T29/OR2T5 antibodies requires careful optimization for successful detection. Based on validated protocols, follow this methodological approach:

Sample Preparation:

• Prepare cell or tissue lysates using a compatible lysis buffer containing protease inhibitors.

• For OR2T29/OR2T5 detection, human cell lines such as HeLa and K562 have been validated as positive controls .

• Determine protein concentration using standard methods (Bradford, BCA).

• Mix samples with loading buffer containing reducing agent and heat at 95°C for 5 minutes.

Gel Electrophoresis and Transfer:

• Load 20-50 μg of protein per lane on an SDS-PAGE gel (10-12% recommended).

• Transfer proteins to a PVDF or nitrocellulose membrane using standard transfer conditions.

Antibody Incubation:

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

• Incubate with primary OR2T29/OR2T5 antibody at appropriate dilution:

  • For monoclonal antibodies (YP-mAb-13513): 1:500-1:2000 dilution .

  • For polyclonal antibodies (STJ94689, A98552): 1:500-1:2000 dilution .

• Incubate overnight at 4°C with gentle agitation.

• Wash membrane 3-5 times with TBST, 5 minutes each.

• Incubate with appropriate HRP-conjugated secondary antibody (anti-mouse or anti-rabbit, depending on primary antibody).

• Wash 3-5 times with TBST, 5 minutes each.

Detection:

• Apply chemiluminescent substrate to the membrane.

• Expose to X-ray film or capture images using a digital imaging system.

• Expected molecular weight for OR2T29/OR2T5: 34-35 kDa .

Controls and Validation:

• Include a positive control (e.g., HeLa or K562 cell lysate).

• Use a blocking peptide control by pre-incubating the antibody with the immunizing peptide .

• Include a loading control antibody (e.g., β-actin, GAPDH).

Experimental evidence shows that OR2T29/OR2T5 antibodies have successfully detected endogenous levels of these proteins in HeLa and K562 cell lysates, with the observed band at approximately 34 kDa .

What are the optimal conditions for using OR2T29/OR2T5 antibodies in immunofluorescence studies?

Immunofluorescence (IF) using OR2T29/OR2T5 antibodies allows visualization of the cellular localization of these receptors. Based on validated protocols with MCF7 cells, follow this detailed methodology:

Sample Preparation:

• Grow cells on sterile glass coverslips or chamber slides to 70-80% confluence.

• For tissue sections, prepare frozen or paraffin-embedded sections following standard protocols.

Fixation and Permeabilization:

• Fix cells using 4% paraformaldehyde in PBS for 15-20 minutes at room temperature.

• Wash 3 times with PBS, 5 minutes each.

• Permeabilize with 0.1-0.5% Triton X-100 in PBS for 5-10 minutes.

• Wash 3 times with PBS, 5 minutes each.

Blocking and Antibody Incubation:

• Block with 5% normal serum (from the same species as the secondary antibody) in PBS for 1 hour at room temperature.

• Incubate with primary OR2T29/OR2T5 antibody diluted in blocking solution:

  • Recommended dilution for polyclonal antibodies: 1:200-1:1000 .

• Incubate overnight at 4°C in a humidified chamber.

• Wash 3 times with PBS, 5 minutes each.

• Incubate with fluorophore-conjugated secondary antibody at recommended dilution in blocking solution.

• Incubate for 1-2 hours at room temperature in the dark.

• Wash 3 times with PBS, 5 minutes each.

Counterstaining and Mounting:

• Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes.

• Wash with PBS.

• Mount coverslips using antifade mounting medium.

• Seal edges with nail polish for long-term storage.

Controls and Validation:

• Include a negative control by omitting the primary antibody.

• Use a peptide competition control by pre-incubating the antibody with the immunizing peptide .

• If possible, include a positive control (e.g., cell line with known expression).

Experimental evidence has shown that OR2T29/OR2T5 antibodies can successfully detect these proteins in MCF7 cells using immunofluorescence. The staining pattern is consistent with the expected membrane localization, as these are multi-pass membrane proteins .

How can researchers validate the specificity of OR2T29/OR2T5 antibodies in their experimental systems?

Validating antibody specificity is critical for ensuring reliable research results. For OR2T29/OR2T5 antibodies, employ these complementary approaches:

1. Peptide Competition Assay:

• Pre-incubate the antibody with the synthetic peptide used as the immunogen.

• Use both the blocked and unblocked antibody in parallel experiments.

• Specific signals should be reduced or eliminated in the blocked condition.

• This has been validated for OR2T29/OR2T5 antibodies in both Western blot and immunofluorescence applications .

2. Multiple Antibody Validation:

• Use different antibodies targeting distinct epitopes of OR2T29/OR2T5.

• Concordant results with multiple antibodies increase confidence in specificity.

• Compare results from both monoclonal and polyclonal antibodies when possible .

3. Genetic Validation:

• Use cells with genetic manipulation of OR2T29/OR2T5 expression:

  • Knockdown by siRNA or shRNA

  • Knockout using CRISPR-Cas9

  • Overexpression systems

• Compare antibody signals in modified vs. control cells.

4. Cross-Reactivity Assessment:

• Test antibody on samples from multiple species to confirm the expected reactivity profile.

• Most OR2T29/OR2T5 antibodies react with human, rat, and mouse samples .

• Examine potential cross-reactivity with other closely related olfactory receptors.

5. Mass Spectrometry Validation:

• Perform immunoprecipitation using the antibody.

• Analyze the precipitated proteins by mass spectrometry.

• Confirm the presence of OR2T29/OR2T5 peptides in the results.

Experimental Evidence:
Data from validated experiments show that OR2T29/OR2T5 antibodies detect a protein of the expected molecular weight (34-35 kDa) in Western blot analyses. Peptide competition assays have demonstrated specificity, as shown in experiments with MCF7 and K562 cells, where the signal was successfully blocked by pre-incubation with the synthesized peptide .

What factors influence the storage and handling of OR2T29/OR2T5 antibodies, and how do these affect experimental outcomes?

Proper storage and handling of OR2T29/OR2T5 antibodies are essential for maintaining their activity and specificity over time. Follow these guidelines based on manufacturer recommendations and best practices:

Storage Conditions:

• Store antibodies at -20°C for long-term storage (up to 1 year from receipt) .

• For antibodies in solution, store in small aliquots to minimize freeze-thaw cycles.

• Most commercial OR2T29/OR2T5 antibodies are supplied in PBS containing:

  • 50% glycerol (acts as a cryoprotectant)

  • 0.5% BSA (stabilizes the antibody)

  • 0.02% sodium azide (prevents microbial growth) .

Handling Best Practices:

• Upon receipt, briefly centrifuge the vial to ensure all liquid is at the bottom .

• Prepare working aliquots in appropriate volumes for typical experiments.

• Return antibodies to -20°C immediately after use.

• Always use clean pipette tips to avoid contamination.

• Never vortex antibodies; instead, mix gently by inverting or flicking the tube.

• Allow frozen antibodies to thaw completely at cold temperatures (on ice or at 4°C).

• Avoid multiple freeze-thaw cycles, which can lead to:

  • Protein denaturation

  • Formation of protein aggregates

  • Reduced binding efficiency

  • Increased background in experiments .

Working Dilution Preparation:

• Prepare working dilutions fresh before each experiment when possible.

• If working dilutions must be stored, keep at 4°C for no more than 1-2 weeks.

• Include the same preservatives as in the original formulation (e.g., sodium azide).

Experimental Outcome Effects:

• Antibody degradation can lead to reduced signal intensity in all applications.

• Aggregated antibodies may cause increased background and non-specific binding.

• Microbial contamination can degrade antibodies and introduce artifacts.

• Batch-to-batch variation can affect reproducibility, particularly with polyclonal antibodies.

Careful attention to storage and handling will help ensure consistent results across experiments and maximize the lifespan of valuable OR2T29/OR2T5 antibodies.

How do recommended dilution factors for OR2T29/OR2T5 antibodies vary across different experimental applications?

Optimal dilution factors for OR2T29/OR2T5 antibodies vary significantly depending on the application, antibody type, and experimental system. Here's a comprehensive comparison based on experimental validation:

Application-Specific Considerations:

Western Blot:

• Higher antibody concentrations (1:500) may be needed for proteins expressed at low levels or when using less sensitive detection methods.

• More dilute antibody solutions (1:2000) are suitable for abundantly expressed proteins or when using highly sensitive detection systems.

• Signal-to-noise ratio should be optimized through titration experiments.

Immunofluorescence:

• More concentrated antibody solutions (1:200) are typically required compared to Western blot.

• Fixation method significantly impacts epitope accessibility and optimal dilution.

• Background fluorescence must be carefully controlled through proper blocking and dilution.

ELISA:

• Requires much higher dilutions (1:10000-1:20000) due to the high sensitivity of the detection system.

• Direct coating vs. sandwich ELISA formats may require different dilutions.

• Optimization through checkerboard titration is recommended.

Immunohistochemistry:

• Typically requires more concentrated antibody solutions compared to other applications.

• Antigen retrieval methods strongly influence optimal dilution.

• Tissue-specific factors may necessitate adjustments to standard dilutions.

When working with new experimental systems or antibody lots, performing a dilution series is strongly recommended to determine the optimal working concentration. The goal is to achieve specific staining with minimal background, maximizing the signal-to-noise ratio.

What are common issues when using OR2T29/OR2T5 antibodies in Western Blot, and how can they be systematically resolved?

When using OR2T29/OR2T5 antibodies in Western blot experiments, researchers may encounter several challenges. Here are common issues and methodological solutions:

1. Weak or No Signal:

Potential Causes:

• Insufficient protein loading

• Inefficient protein transfer

• Low antibody concentration

• Degraded antibody

• Low expression of target protein

Systematic Solutions:

• Increase protein loading (50-80 μg per lane)

• Optimize transfer conditions (consider longer transfer time or semi-dry transfer)

• Use more concentrated primary antibody (1:500 dilution instead of 1:2000)

• Extend primary antibody incubation time (overnight at 4°C)

• Use fresh antibody aliquot

• Enhance detection using more sensitive substrates (e.g., femto vs. pico chemiluminescent substrate)

• Verify expression of OR2T29/OR2T5 in your sample (use validated positive controls like HeLa or K562 cells)

2. High Background:

Potential Causes:

• Insufficient blocking

• Too concentrated antibody

• Inadequate washing

• Cross-reactivity

Systematic Solutions:

• Extend blocking time (2-3 hours at room temperature)

• Try different blocking agents (5% BSA may work better than milk for some applications)

• Dilute primary antibody further (1:1000-1:2000)

• Increase number and duration of wash steps

• Add 0.05-0.1% Tween-20 to antibody dilution buffer

• Use different secondary antibody

3. Multiple Bands or Unexpected Band Size:

Potential Causes:

• Cross-reactivity with related proteins

• Protein degradation

• Post-translational modifications

• Splice variants

• Non-specific binding

Systematic Solutions:

• Verify expected molecular weight (34-35 kDa for OR2T29/OR2T5)

• Add fresh protease inhibitors to lysis buffer

• Use peptide competition assay to identify specific bands

• Try reducing SDS concentration in running buffer

• Prepare fresh samples

• Use freshly prepared gels

4. Inconsistent Results Between Experiments:

Potential Causes:

• Variation in transfer efficiency

• Inconsistent sample preparation

• Antibody degradation

• Variation in cell culture conditions

Systematic Solutions:

• Include loading controls (β-actin, GAPDH)

• Standardize protein concentration determination method

• Use consistent sample preparation protocols

• Aliquot antibodies to avoid multiple freeze-thaw cycles

• Document lot numbers and experimental conditions

Optimized Protocol Based on Experimental Evidence:
Based on validated experiments with OR2T29/OR2T5 antibodies, successful detection has been achieved using:

• HeLa and K562 cell lysates as positive controls

• 1:500-1:1000 dilution of primary antibody

• Overnight incubation at 4°C

• Standard ECL detection systems

• Expected band size of approximately 34 kDa

Peptide competition assays have been particularly useful for validating signal specificity, as demonstrated in experimental data where pre-incubation with the synthesized peptide effectively blocked the signal in Western blot analysis of K562 cell lysates .

How can researchers systematically address non-specific binding and high background issues in immunofluorescence studies with OR2T29/OR2T5 antibodies?

Immunofluorescence studies with OR2T29/OR2T5 antibodies may present specific challenges related to background and specificity. Here's a systematic approach to resolving these issues:

1. Detailed Background Reduction Protocol:

Step-by-Step Methodology:

• Optimize fixation:

  • Compare different fixatives (4% PFA, methanol, acetone)

  • Limit fixation time to minimize epitope masking

  • Ensure complete permeabilization (0.1-0.3% Triton X-100 for 5-10 minutes)

• Enhance blocking:

  • Use 5-10% serum from the species of the secondary antibody

  • Add 1% BSA to reduce non-specific binding

  • Consider specialized blocking reagents for problematic samples

  • Extend blocking time to 2 hours at room temperature

• Antibody optimization:

  • Titrate antibody concentration (try 1:500 instead of 1:200)

  • Prepare antibodies in blocking solution

  • Incubate at 4°C overnight rather than at room temperature

  • Centrifuge diluted antibody (12,000 × g for 10 minutes) before use to remove aggregates

• Intensify washing:

  • Use PBS with 0.05-0.1% Tween-20

  • Increase wash duration (5 × 10 minutes)

  • Use gentle agitation during washing

  • Perform PBS rinses before and after primary and secondary antibody incubations

• Reduce autofluorescence:

  • Treat with 0.1% Sudan Black B in 70% ethanol (5 minutes)

  • Use freshly prepared 0.1% sodium borohydride in PBS (10 minutes)

  • Include 10 mM NH4Cl in wash buffer

2. Systematic Control Experiments for Specificity Validation:

Essential Controls:

• Primary antibody controls:

  • Omit primary antibody (secondary only control)

  • Use isotype control antibody at the same concentration

  • Pre-absorb antibody with immunizing peptide

  • Compare multiple antibodies against different epitopes

• Cell/tissue controls:

  • Use known positive cell lines (MCF7 has been validated)

  • Include negative control cells (if available)

  • Compare normal vs. OR2T29/OR2T5 overexpressing cells

• Fluorophore controls:

  • Include single-label controls when performing multi-label experiments

  • Evaluate spectral bleed-through

  • Use appropriate filter sets to minimize cross-talk

• Image acquisition controls:

  • Set exposure parameters using secondary-only controls

  • Maintain identical acquisition settings across all samples

  • Use sequential scanning for multi-channel imaging

3. Advanced Troubleshooting for Persistent Issues:

For Membrane Protein-Specific Challenges:

• Gentle fixation approaches:

  • Use lower concentration fixatives (2% PFA)

  • Reduce fixation time (10 minutes)

  • Try combinatorial approaches (0.5% PFA with 0.1% glutaraldehyde)

• Alternative permeabilization methods:

  • Use milder detergents (0.01-0.05% saponin)

  • Try digitonin for selective plasma membrane permeabilization

  • Consider freeze-thaw permeabilization for sensitive epitopes

• Signal amplification strategies:

  • Apply tyramide signal amplification for low-abundance targets

  • Use biotinylated secondary antibodies with streptavidin-fluorophore conjugates

  • Consider QDot-conjugated antibodies for improved sensitivity and photostability

Successful immunofluorescence detection of OR2T29/OR2T5 has been documented in MCF7 cells, showing membrane and cytoplasmic distribution consistent with the expected localization of these multi-pass membrane proteins . Peptide competition experiments provide the most reliable method for confirming antibody specificity in immunofluorescence applications.

How can OR2T29/OR2T5 antibodies be applied in studies of olfactory signal transduction pathways and receptor trafficking?

OR2T29/OR2T5 antibodies serve as valuable tools for investigating complex olfactory receptor signaling and trafficking mechanisms. Here are advanced methodological approaches:

1. Receptor Trafficking and Localization Studies:

Methodology:

• Dual immunofluorescence labeling with OR2T29/OR2T5 antibodies and organelle markers:

  • Plasma membrane (Na+/K+-ATPase)

  • Endoplasmic reticulum (calnexin)

  • Golgi apparatus (GM130)

  • Endosomes (EEA1, Rab proteins)

• Live-cell imaging using OR2T29/OR2T5 antibodies against extracellular epitopes on non-permeabilized cells

• Immunoelectron microscopy for ultrastructural localization

• FRET/BRET assays to study protein-protein interactions during trafficking

Applications:

• Investigate receptor internalization upon ligand binding

• Study quality control mechanisms in the ER for these multi-pass membrane proteins

• Examine the role of chaperones in receptor folding and transport

• Analyze receptor recycling pathways

2. Co-immunoprecipitation for Protein Interaction Studies:

Methodology:

• Prepare cell lysates under non-denaturing conditions

• Pre-clear lysates with Protein A/G beads

• Immunoprecipitate with OR2T29/OR2T5 antibodies

• Wash extensively to remove non-specific interactions

• Elute bound proteins

• Analyze by Western blot or mass spectrometry

Applications:

• Identify novel binding partners of OR2T29/OR2T5

• Study interaction with G proteins (Gαolf) and other signaling components

• Investigate receptor dimerization or oligomerization

• Map the interactome of olfactory receptors in different cell types

3. Advanced Imaging Techniques:

Methodology:

• Super-resolution microscopy (STORM, PALM, SIM) with OR2T29/OR2T5 antibodies

• Single-molecule tracking of receptor dynamics

• FRAP (Fluorescence Recovery After Photobleaching) for mobility studies

• Expansion microscopy for enhanced spatial resolution

Applications:

• Visualize receptor nano-clusters on the membrane

• Study receptor diffusion dynamics

• Analyze co-localization with signaling components at nanoscale resolution

• Investigate structural changes upon receptor activation

4. Functional Studies Using Antibody-Based Manipulation:

Methodology:

• Use antibodies against extracellular domains for functional blocking experiments

• Develop internalizing antibodies for receptor downregulation

• Create cell-penetrating antibody derivatives to target intracellular domains

Applications:

• Modulate receptor signaling in live cells

• Study the consequences of receptor blockade on downstream pathways

• Develop therapeutic approaches for disorders involving olfactory receptor dysfunction

These advanced applications leverage OR2T29/OR2T5 antibodies to explore the complex biology of olfactory receptors, from their trafficking and signaling to their functional properties in neuronal and non-neuronal contexts.

What potential roles do OR2T29/OR2T5 play in neurological disorders, and how can antibodies facilitate this research?

Recent studies suggest potential roles for olfactory receptors, including OR2T29/OR2T5, in various neurological conditions. Antibodies against these receptors provide critical tools for investigating these connections:

1. Autism Spectrum Disorders (ASD) Research:

Research Context:
Studies indicate that olfactory receptor genes, including OR genes, were found to be enriched in both ASD and non-ASD groups in genome-wide analyses. Specifically, "167 of 990 genes in the nonASD gene-set (Fold Enrichment = 8.35; FDR = 1.88 × 10^-84) were OR genes."

Methodological Approaches:

• Comparative expression analysis:

  • Collect post-mortem brain tissue or patient-derived cell lines

  • Perform immunohistochemistry or Western blot using OR2T29/OR2T5 antibodies

  • Compare expression levels between ASD and control samples

  • Correlate with genetic variation data

• Functional studies in ASD models:

  • Use ASD mouse models or patient-derived iPSCs

  • Manipulate OR2T29/OR2T5 expression (overexpression, knockdown)

  • Assess effects on neuronal development, connectivity, and function

  • Monitor expression using specific antibodies

• Genetic association studies:

  • Analyze copy number variations (CNVs) in OR gene regions

  • Correlate genetic variations with receptor expression using antibodies

  • Investigate functional consequences of variants

2. Primary Cilium Dysfunction Research:

Research Context:
The search results mention genes involved in "primary cilium assembly and organization" being enriched in ASD cohorts . Olfactory receptors are expressed in cilia, suggesting potential connections to ciliopathies.

Methodological Approaches:

• Cilia localization studies:

  • Perform co-immunofluorescence with OR2T29/OR2T5 antibodies and cilia markers

  • Use super-resolution microscopy to visualize receptor distribution in cilia

  • Examine receptor trafficking to cilia in disease models

• Functional assays:

  • Investigate the impact of ciliary dysfunction on OR2T29/OR2T5 signaling

  • Monitor calcium responses in ciliopathy models

  • Verify receptor expression and localization using specific antibodies

3. JAK-STAT Pathway Connections:

Research Context:
The search results mention the JAK-STAT pathway being overrepresented in gene sets with CNVs in neurological disorders . This pathway mediates cellular transcriptional responses to cytokines and is related to immune response.

Methodological Approaches:

• Signaling pathway analysis:

  • Investigate potential crosstalk between OR2T29/OR2T5 and JAK-STAT signaling

  • Perform co-immunoprecipitation with pathway components

  • Analyze phosphorylation status of STAT proteins following receptor activation

  • Use phospho-specific antibodies in combination with OR2T29/OR2T5 antibodies

• Inflammatory response studies:

  • Examine receptor expression changes during neuroinflammation

  • Investigate receptor functions in immune cells

  • Assess the impact of cytokine signaling on receptor expression and function

4. Olfactory Dysfunction in Neurological Disorders:

Methodological Approaches:

• Diagnostic applications:

  • Develop immunohistochemical protocols for OR2T29/OR2T5 in olfactory epithelium biopsies

  • Correlate receptor alterations with clinical olfactory testing

  • Explore potential diagnostic value in early disease detection

• Therapeutic target identification:

  • Screen for compounds modulating receptor function

  • Verify compound effects on receptor expression and localization using antibodies

  • Develop targeted delivery systems to restore receptor function

OR2T29/OR2T5 antibodies enable precise detection and quantification of these receptors in various experimental systems, from cell culture to animal models and human samples. Their specificity is crucial for differentiating between closely related olfactory receptors and for validating genetic findings at the protein level.