NR1I3 Antibody

What is NR1I3 and why is it significant in research?

NR1I3 (Nuclear Receptor Subfamily 1 Group I Member 3) is a member of the nuclear steroid/thyroid hormone receptor superfamily. In humans, the canonical protein consists of 352 amino acid residues with a molecular mass of 39.9 kDa. NR1I3 is primarily localized in the nucleus and cytoplasm, with up to 15 different isoforms reported. It is highly expressed in the liver and functions by binding and transactivating retinoic acid response elements that control expression of the retinoic acid receptor beta 2 and alcohol dehydrogenase 3 genes . Its significance in research stems from its role in xenobiotic metabolism, making it crucial for studies related to drug metabolism, liver function, and toxicology research.

What are the common synonyms and alternative names for NR1I3?

Researchers should be aware of multiple nomenclatures when searching literature for NR1I3. Common synonyms include:

• CAR1

• MB67

• Constitutive Activator of Retinoid Response

• Constitutive Active Receptor

• Constitutive Active Response

• CAR (Constitutive Androstane Receptor)

Understanding these alternative designations is essential when conducting literature searches or ordering antibodies, as different suppliers and research groups may use varying terminology.

In which species has NR1I3 been characterized, and how conserved is it?

NR1I3 gene orthologs have been reported in multiple species including:

• Mouse

• Rat

• Bovine

• Frog

• Chimpanzee

• Chicken

The conservation across species makes NR1I3 valuable for comparative studies and allows for translational research between animal models and human applications. When selecting antibodies for cross-species applications, researchers should verify the epitope conservation and validated reactivity across target species.

What criteria should be considered when selecting an NR1I3 antibody for specific applications?

When selecting an NR1I3 antibody, researchers should consider:

• Application compatibility: Different antibodies are optimized for specific applications such as Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), ChIP, or ELISA. For example, multiple suppliers offer NR1I3 antibodies validated for Western Blot applications, while fewer are optimized for ChIP studies .

• Species reactivity: Ensure the antibody reacts with your species of interest. Available antibodies show reactivity with human, mouse, rat, and other species depending on the product .

• Epitope location: Some antibodies target the C-terminal region or other specific domains, which may affect detection of particular isoforms or mutated variants .

• Validation data: Priority should be given to antibodies with published citations or comprehensive validation data demonstrating specificity.

• Antibody format: Consider whether native, conjugated, or tagged formats are most appropriate for your experimental design.

How can I validate the specificity of an NR1I3 antibody?

To validate NR1I3 antibody specificity, implement the following methodological approach:

• Positive and negative controls:

  • Use tissue known to express high levels of NR1I3 (e.g., liver) as a positive control

  • Use tissue with minimal expression or knockout models (Car−/−) as negative controls

• Knockdown validation: Compare antibody reactivity in wild-type versus NR1I3 knockdown or knockout samples

• Multiple antibody verification: Test multiple antibodies targeting different epitopes of NR1I3

• Molecular weight confirmation: Verify that the detected band corresponds to the expected molecular weight (approximately 39.9 kDa for the canonical human form)

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm signal elimination

What are the optimal protocols for using NR1I3 antibodies in Western blot applications?

For optimal Western blot results with NR1I3 antibodies:

• Sample preparation:

  • Prepare nuclear extracts to enrich for NR1I3, which is primarily localized in the nucleus

  • Use a standard protocol for nuclear extraction as described in previous literature

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal resolution of the 39.9 kDa NR1I3 protein

  • Load 20-50 μg of total protein per lane depending on expression levels

• Transfer and blocking:

  • Transfer to PVDF or nitrocellulose membranes

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

• Antibody incubation:

  • Dilute primary antibody according to manufacturer's recommendations (typically 1:500-1:1000)

  • Incubate overnight at 4°C

  • Wash thoroughly with TBST

  • Use appropriate HRP-conjugated secondary antibody (typically 1:2000-1:5000)

• Detection:

  • Use enhanced chemiluminescence (ECL) detection system

  • Expose to X-ray film or use digital imaging systems

What are the recommended protocols for immunohistochemical detection of NR1I3?

For immunohistochemical detection of NR1I3:

• Tissue preparation:

  • Fix tissue in 10% formalin

  • Process and embed in paraffin

  • Section at 4-5 μm thickness

• Antigen retrieval:

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Heat in pressure cooker or microwave for 15-20 minutes

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% H₂O₂

  • Block non-specific binding with serum

  • Incubate with primary NR1I3 antibody (dilution typically 1:100-1:200) overnight at 4°C

  • Use the avidin-biotin-peroxidase complex method for detection

• Visualization:

  • Develop with DAB substrate

  • Counterstain with hematoxylin

  • Dehydrate, clear, and mount

• Controls:

  • Include positive controls (liver tissue) and negative controls (omitting primary antibody)

  • Consider using Car−/− tissue as a specificity control when available

How can I optimize NR1I3 detection in difficult tissue samples?

For challenging tissue samples:

• Optimize fixation time: Excessive fixation can mask epitopes; consider using freshly fixed tissues or optimizing fixation duration

• Antigen retrieval optimization:

  • Test multiple antigen retrieval methods (heat, enzymatic, pH variations)

  • Extend antigen retrieval time for difficult samples

• Signal amplification strategies:

  • Implement tyramide signal amplification

  • Use polymer-based detection systems for enhanced sensitivity

• Antibody concentration optimization:

  • Perform titration experiments to determine optimal antibody concentration

  • Consider longer incubation times at lower concentrations

• Background reduction:

  • Use specialized blocking reagents to reduce non-specific binding

  • Include additives like Triton X-100 for improved penetration in tissue sections

  • Apply avidin/biotin blocking for tissues with high endogenous biotin

How can NR1I3 antibodies be utilized in studying zone-specific liver gene expression?

NR1I3 exhibits zone-specific expression patterns in the liver. For investigating zone-specific expression:

• Laser Capture Microdissection (LCM) approach:

  • Prepare frozen liver tissue sections and stain with 1% Cresyl Violet acetate

  • Identify and capture periportal (Zone 1) or centrilobular (Zone 3) areas using laser capture microscopy

  • Collect 40-60 regions (approximately 0.03-0.04 mm² each)

  • Extract RNA using specialized kits designed for LCM samples

  • Validate zonal separation by examining zone-specific marker expression

• Immunofluorescence co-localization:

  • Use NR1I3 antibodies in combination with zone-specific markers

  • Perform confocal microscopy to analyze co-localization patterns

  • Quantify signal intensity across different zones

• Analysis parameters:

  • Measure relative expression levels between zones

  • Correlate with target gene expression

  • Evaluate changes in zonal expression under different physiological or pathological conditions

What strategies can be employed to investigate NR1I3 involvement in liver injury and regeneration?

To investigate NR1I3's role in liver injury and regeneration:

• Comparative analysis of wild-type and knockout models:

  • Utilize Car+/+ and Car−/− mice to study differential responses to liver injury

  • Feed experimental diet (e.g., DDC diet) to induce liver injury

  • Assess liver injury through histological analysis and biochemical markers

• Ductular reaction assessment:

  • Perform immunohistochemistry using A6 antibody (oval cell marker)

  • Quantify ductular reaction in periportal areas

  • Compare between wild-type and Car−/− mice

• Proliferation and apoptosis analysis:

  • Use PCNA antibody to assess hepatocyte proliferation

  • Apply TUNEL assay for apoptosis detection

  • Count positive cells in multiple fields (recommended: 9-12 periportal or centrilobular areas at 200× magnification)

• Gene expression analysis:

  • Extract RNA from liver tissue or zone-specific samples

  • Perform real-time PCR using TaqMan or SYBR Green methods

  • Analyze expression of NR1I3 target genes involved in regeneration

How can I investigate NR1I3 protein-protein interactions and transcriptional complexes?

For studying NR1I3 protein interactions and transcriptional activity:

• Co-immunoprecipitation (Co-IP) approach:

  • Use NR1I3 antibodies to pull down protein complexes

  • Identify interacting partners via Western blot or mass spectrometry

  • Compare interaction patterns under different physiological conditions

• Chromatin Immunoprecipitation (ChIP) methodology:

  • Use ChIP-grade NR1I3 antibodies to isolate protein-DNA complexes

  • Design primers targeting known or putative NR1I3 binding sites

  • Perform quantitative PCR or sequencing to identify binding regions

  • Consider using reporter constructs like (NR1)5-tk-luciferase to assess transcriptional activity

• Proximity ligation assay (PLA):

  • Detect protein-protein interactions in situ

  • Use NR1I3 antibody in combination with antibodies against suspected interaction partners

  • Visualize interactions as fluorescent spots within cells

What are common issues encountered when using NR1I3 antibodies and how can they be resolved?

How can I quantify NR1I3 expression levels in tissue samples for comparative studies?

For accurate quantification of NR1I3 expression:

• Immunohistochemical quantification:

  • Use consistent staining protocols across all samples

  • Capture images at identical exposure settings

  • Apply software analysis (ImageJ, QuPath) for:

    • H-score calculation (staining intensity × percentage of positive cells)

    • Automated cell counting of positive vs. negative cells

    • Subcellular localization assessment (nuclear vs. cytoplasmic)

  • Count in multiple fields (recommended: at least 9-12 areas at 200× magnification)

• Western blot quantification:

  • Include loading controls (β-actin for cytoplasmic, Lamin B for nuclear fractions)

  • Use standard curves with recombinant protein

  • Apply densitometry analysis and normalize to loading controls

  • Present data as relative expression compared to controls

• RT-qPCR analysis:

  • Design primers specific to NR1I3 (avoid regions with high homology to other nuclear receptors)

  • Use appropriate reference genes (GAPDH, β-actin, or tissue-specific stable references)

  • Apply the ΔΔCt method for relative quantification

  • Consider using TaqMan probes for enhanced specificity

What are the best approaches for studying NR1I3 in primary hepatocytes versus cell lines?

When working with primary hepatocytes:

• Isolate using established collagenase perfusion protocols

• Maintain in appropriate media supplemented with dexamethasone to stabilize phenotype

• Use within 24-48 hours of isolation for optimal NR1I3 expression levels

• Consider sandwich culture models for extended studies