OR10X1 Antibody

Basic Research Questions

• What is OR10X1 protein and what are its key structural characteristics?

  OR10X1 (olfactory receptor family 10 subfamily X member 1) is a protein belonging to the G-protein coupled receptor 1 family. In humans, the canonical protein consists of 326 amino acid residues with a molecular mass of approximately 36.4 kDa. It features a characteristic 7-transmembrane domain structure common to olfactory receptors and is primarily localized in the cell membrane. OR10X1 undergoes post-translational modifications, notably glycosylation. The protein is also known by several synonyms including olfactory receptor OR1-13 pseudogene, olfactory receptor OR1-14, and olfactory receptor 10X1. Orthologs have been identified in mouse and chimpanzee species .

• What are the common applications for OR10X1 antibodies in research?

  OR10X1 antibodies are primarily employed in several immunodetection techniques. Western Blot (WB) is the most commonly utilized application for detecting OR10X1 protein expression in cell or tissue lysates. ELISA represents another frequent application for quantitative measurement of OR10X1 levels. Immunofluorescence (IF) and immunocytochemistry (ICC) are valuable for visualizing the subcellular localization of OR10X1 within intact cells or tissues. These applications provide researchers with complementary approaches to investigate OR10X1 expression, localization, and function in various experimental contexts .

• What species reactivity is available for commercial OR10X1 antibodies?

  Commercial OR10X1 antibodies demonstrate varying species reactivity profiles. Many are specifically developed for human OR10X1 detection, while others offer cross-reactivity with additional species. Based on available products, antibodies with the following reactivity patterns can be found:

  Reactivity Pattern	Available Applications	Host
  Human only	WB, ELISA, IF	Rabbit
  Human, Mouse	WB, ELISA	Rabbit
  Human, Mouse, Rat	WB, IF, ELISA	Rabbit
  Human, Rat	WB	Rabbit
  Human, Mouse, Rat, Cow, Dog, Horse, Pig, Rabbit	WB	Rabbit

  When selecting an OR10X1 antibody for your research, it's essential to verify the specific reactivity profile for your species of interest .

Antibody Characteristics and Selection

• What are the differences between monoclonal and polyclonal OR10X1 antibodies?

  While most commercially available OR10X1 antibodies are polyclonal (typically rabbit-derived), the choice between polyclonal and monoclonal antibodies depends on your specific research requirements:

  Polyclonal OR10X1 antibodies:

  • Recognize multiple epitopes on the OR10X1 protein

  • Generally provide higher sensitivity due to multiple epitope binding

  • Show greater tolerance to minor protein denaturation or modifications

  • Useful for detecting proteins expressed at low levels

  • Most OR10X1 commercial antibodies are polyclonal raised in rabbits

  Monoclonal OR10X1 antibodies:

  • Target a single epitope with high specificity

  • Offer greater consistency between batches

  • Reduce background in some applications

  • May be less sensitive than polyclonal antibodies

  For initial characterization studies of OR10X1, polyclonal antibodies may provide better sensitivity, while monoclonal antibodies might be preferred for highly specific detection of particular OR10X1 epitopes or conformations .

• What epitope regions are targeted by different OR10X1 antibodies?

  Commercial OR10X1 antibodies target various regions of the protein, each offering different advantages depending on experimental needs:

  Epitope Region	Amino Acid Range	Applications	Notes
  C-terminal region	Not specified	WB, ELISA, IF	Detects endogenous levels of OR10X1
  Middle region	216-265	WB, IF	Used for detection in human samples
  N-terminal region	83-112	WB	Available for human and mouse samples
  Internal region	Not specified	WB, ELISA, IF, ICC	Multiple applications possible

  When selecting an OR10X1 antibody, consider whether your experimental conditions might affect epitope accessibility (e.g., protein folding, post-translational modifications, or protein-protein interactions). C-terminal antibodies often work well for Western blot applications, while antibodies targeting extracellular domains may be preferable for flow cytometry or immunoprecipitation .

• How should OR10X1 antibodies be stored and handled to maintain optimal activity?

  Proper storage and handling of OR10X1 antibodies is crucial for maintaining their activity and specificity:

  • Storage temperature: Store at -20°C for long-term storage (one year or more). For frequent use within a month, 4°C storage is acceptable.

  • Avoid repeated freeze-thaw cycles that can degrade antibody quality. Aliquot antibodies into smaller volumes upon initial thawing.

  • Most OR10X1 antibodies are supplied in a stabilizing solution containing 50% glycerol and 0.02% sodium azide in PBS.

  • When diluting the antibody for experiments, use fresh, high-quality buffer systems recommended for each specific application.

  • Always centrifuge the antibody vial briefly before opening to collect all liquid at the bottom.

  • For long-term storage of diluted antibodies, add a carrier protein such as BSA (0.1-1%) to prevent adsorption to container surfaces and maintain stability .

Experimental Methodology

• What is the recommended protocol for OR10X1 Western blot analysis?

  For optimal Western blot detection of OR10X1:

  1. Sample preparation:

     • Extract proteins from tissues or cells using an appropriate lysis buffer with protease inhibitors

     • Denature samples at 95°C for 5 minutes in sample buffer containing SDS and reducing agent

  2. Gel electrophoresis and transfer:

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

     • Transfer proteins to PVDF or nitrocellulose membrane

  3. Blocking and antibody incubation:

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

     • Incubate with primary OR10X1 antibody at 1:500-1:2000 dilution in blocking buffer overnight at 4°C

     • Wash 3-5 times with TBST

     • Incubate with appropriate HRP-conjugated secondary antibody at 1:2000-1:5000 dilution for 1 hour at room temperature

     • Wash 3-5 times with TBST

  4. Detection:

     • Develop using ECL substrate and appropriate imaging system

     • Expected molecular weight for OR10X1 is approximately 36.4 kDa

  5. Validation controls:

     • Include positive control samples known to express OR10X1

     • Consider using blocking peptide controls to confirm specificity

• What are the optimal conditions for OR10X1 immunofluorescence staining?

  For successful immunofluorescence detection of OR10X1:

  1. Sample preparation:

     • Fix cells with 4% paraformaldehyde for 15-20 minutes at room temperature

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

  2. Blocking and antibody incubation:

     • Block with 1-5% BSA or normal serum in PBS for 30-60 minutes

     • Incubate with primary OR10X1 antibody at 1:100-1:500 dilution overnight at 4°C

     • Wash 3 times with PBS

     • Incubate with fluorophore-conjugated secondary antibody at 1:200-1:1000 dilution for 1 hour at room temperature in the dark

     • Wash 3 times with PBS

  3. Counterstaining and mounting:

     • Counterstain nuclei with DAPI

     • Mount using an appropriate anti-fade mounting medium

  4. Imaging considerations:

     • OR10X1 should show primarily membrane localization

     • Include proper controls including secondary-only controls to assess background

     • For co-localization studies, consider using markers for cell membrane, endoplasmic reticulum, or Golgi apparatus

• How can I optimize OR10X1 antibody concentration for different applications?

  Optimizing OR10X1 antibody concentration is crucial for obtaining specific signals while minimizing background:

  1. Titration experiment design:

     • For Western blot: Test dilutions ranging from 1:500 to 1:3000

     • For immunofluorescence: Test dilutions from 1:100 to 1:500

     • For ELISA: Test dilutions from 1:5000 to 1:20000

  2. Optimization approach:

     • Begin with the manufacturer's recommended dilution range

     • Prepare a positive control sample known to express OR10X1

     • Perform parallel experiments with different antibody concentrations

     • Evaluate signal-to-noise ratio for each concentration

  3. Factors affecting optimal concentration:

     • Expression level of OR10X1 in your specific samples

     • Sample preparation method

     • Blocking reagents used

     • Incubation time and temperature

     • Detection system sensitivity

  4. Final optimization:

     • Select the dilution that provides specific signal with minimal background

     • For weak signals, consider longer incubation times rather than higher concentrations

     • Document optimized conditions for reproducibility

• What conjugation options are available for OR10X1 antibodies?

  Various conjugation options are available for OR10X1 antibodies to suit different experimental needs:

  Conjugate Category	Available Options
  Fluorescent proteins	AF350, AF488, AF555, AF594, AF647, AF680, AF700, AF750
  Enzymes	HRP, Alkaline Phosphatase
  Binding proteins	Streptavidin
  Fluorescent tandems	APC, PE and their combinations with Cy5, Cy7, AF610, AF700
  Small molecules	Biotin
  Traditional dyes	FITC, TRITC, PacBlue, PacOrange, Cy3, Cy5
  Modern fluorophores	iFluor and mFluor series covering UV to near-IR spectrum

  Custom conjugation services allow researchers to select the optimal label for their specific experimental setup. When choosing a conjugate, consider factors such as the detection method, potential spectral overlap with other fluorophores in multiplexed experiments, and the sensitivity requirements of your application .

Advanced Research Considerations

• How can I validate the specificity of OR10X1 antibodies?

  Validating OR10X1 antibody specificity is essential for obtaining reliable research results:

  1. Positive and negative controls:

     • Use cell lines or tissues known to express or lack OR10X1

     • Consider overexpression systems with tagged OR10X1 constructs

     • Use siRNA or CRISPR knockout models to create negative controls

  2. Blocking peptide competition:

     • Pre-incubate OR10X1 antibody with its immunizing peptide

     • Compare staining patterns with and without peptide competition

     • Specific signals should be significantly reduced or eliminated

  3. Multiple antibody verification:

     • Use different antibodies targeting distinct OR10X1 epitopes

     • Consistent staining patterns across antibodies increase confidence

  4. Orthogonal methods:

     • Correlate protein detection with mRNA expression (RT-PCR or RNA-seq)

     • Compare results across different detection methods (WB, IF, IHC)

  5. Cross-reactivity assessment:

     • Test in samples expressing closely related olfactory receptors

     • Verify specificity against other OR10 family members

• What is known about OR10X1 expression patterns in different tissues?

  OR10X1 expression follows specific patterns across tissues:

  1. Primary expression:

     • Primarily expressed in olfactory epithelium, consistent with its role in olfactory perception

     • Detected in specialized sensory neurons within the nasal cavity

  2. Additional tissue expression:

     • Emerging evidence suggests expression in some non-olfactory tissues

     • Potential expression in specific cancer types, as OR10X1 has been identified in gastric cancer genetics studies

  3. Expression in disease conditions:

     • OR10X1 has been identified as a gene of interest in gastric cancer studies

     • It appears in a list of significantly altered genes in cancer patients

     • Co-occurrence with other genes like TNFRSF4, MOGS, KMT2C, and FCGR2A has been observed in cancer genetics

     • The gene shows differential alteration rates between age groups in cancer patients (4% prevalence in patients <67 years old)

  4. Experimental considerations:

     • When studying OR10X1, include appropriate tissue controls

     • Consider developmental stage and disease state in expression analysis

• What considerations should be made when using OR10X1 antibodies in multiplexed immunoassays?

  For successful multiplexed immunodetection with OR10X1 antibodies:

  1. Antibody compatibility:

     • Select OR10X1 antibodies raised in different host species than other primary antibodies

     • If using multiple rabbit antibodies, consider directly conjugated versions

     • Verify that primary antibodies don't cross-react with non-target proteins

  2. Signal separation strategies:

     • Choose fluorophores with minimal spectral overlap

     • Consider sequential rather than simultaneous detection for challenging combinations

     • Implement appropriate compensation controls for flow cytometry applications

  3. Epitope accessibility concerns:

     • Verify that multiplexing doesn't interfere with OR10X1 epitope accessibility

     • Test antibody combinations individually before multiplexing

     • Consider the order of antibody application for sequential protocols

  4. Tissue autofluorescence management:

     • Implement appropriate blocking of endogenous peroxidases and biotin

     • Use autofluorescence reduction treatments when necessary

     • Select fluorophores that emit away from tissue autofluorescence spectra

  5. Controls for multiplexed assays:

     • Include single-stained controls for each antibody

     • Use isotype controls to assess non-specific binding

     • Add absorption controls with immunizing peptides when available

• How can I troubleshoot common issues with OR10X1 antibody applications?

  Addressing common challenges when working with OR10X1 antibodies:

  1. No signal in Western blot:

     • Verify OR10X1 expression in your sample type

     • Increase antibody concentration or extend incubation time

     • Consider non-reducing conditions if epitope is conformation-dependent

     • Ensure transfer efficiency with reversible protein stain

     • Test different extraction methods to improve protein solubilization

  2. Multiple bands in Western blot:

     • Verify if bands represent glycosylated forms of OR10X1

     • Check for potential proteolytic degradation by adding fresh protease inhibitors

     • Test antibody specificity with blocking peptide

     • Optimize primary antibody concentration to reduce non-specific binding

  3. High background in immunofluorescence:

     • Increase blocking time or concentration

     • Reduce primary antibody concentration

     • Include additional washing steps

     • Test different fixation methods

     • Use more specific secondary antibodies

  4. Weak or inconsistent staining:

     • Optimize antigen retrieval methods for fixed tissues

     • Extend primary antibody incubation time

     • Verify antibody stability and storage conditions

     • Test different detection systems with higher sensitivity

     • Consider signal amplification methods

• What role does OR10X1 play in genetic studies and how can antibodies contribute to this research?

  OR10X1 has emerging significance in genetic studies with antibody applications:

  1. Cancer genetics implications:

     • OR10X1 appears in genetic studies of gastric cancer

     • It's among genes showing altered expression in cancer patients

     • Shows co-occurrence patterns with other genes like FCGR2A, TNFRSF4, and KMT2C

     • 4% prevalence of alteration in patients under 67 years old

  2. Antibody applications in genetic research:

     • Validate genetic findings at protein expression level

     • Study the consequences of identified mutations on protein localization

     • Assess correlation between genetic alterations and protein expression

     • Investigate protein-protein interactions affected by genetic variants

  3. Methodological approaches:

     • Use OR10X1 antibodies to validate results from genomic and transcriptomic studies

     • Compare protein expression between wild-type and mutant forms

     • Employ proximity ligation assays to study altered protein interactions

     • Implement immunoprecipitation to identify binding partners

  4. Future research directions:

     • Investigate functional consequences of OR10X1 mutations

     • Study potential non-olfactory roles in disease contexts

     • Explore application of OR10X1 as a potential biomarker

     • Examine relationship with signaling pathways implicated in disease