NR1I2 Antibody

What is NR1I2/PXR and what are its main functions in biological systems?

NR1I2/PXR is a member of the nuclear receptor superfamily that plays a key role in the induction of genes involved in drug transport and metabolism. It modulates drug transport and metabolism through the regulation of target genes responsible for the transport and conversion of chemicals into metabolites that are more easily eliminated from the body . Additionally, it serves as an endobiotic receptor that regulates inflammatory responses and functions as a transcription factor that activates multiple genes involved in the metabolism and secretion of potentially harmful xenobiotics, drugs, and endogenous compounds . NR1I2 forms heterodimers with Retinoid X Receptor (RXR) and is activated by various compounds including pregnanolone, progesterone, xenobiotics, and endobiotics .

What are the different isoforms of NR1I2 and how do they affect antibody selection?

NR1I2 exists in at least seven splice forms from a single gene. Two primary forms are PXR1 (434 amino acids) and PXR2 (473 amino acids), with the latter having a 39 amino acid extension at the N-terminus resulting from the use of an alternate exon 1 . When selecting antibodies, researchers should consider which isoform(s) they need to detect and choose antibodies with appropriate epitope recognition. For instance, antibodies targeting the N-terminal region may differentiate between PXR1 and PXR2, while those targeting conserved regions will detect multiple isoforms.

What are the validated applications for NR1I2 antibodies?

NR1I2 antibodies have been validated for multiple experimental applications including:

Different antibodies may have varying efficacy across applications, so researchers should verify validation data for their specific experimental needs .

How should I optimize Western blot conditions for detecting NR1I2?

For optimal Western blot detection of NR1I2:

• Sample preparation: Use appropriate lysis buffers with protease inhibitors. Nuclear extraction protocols may improve yield as NR1I2 is a nuclear receptor.

• Protein loading: Load adequate protein (typically 20-50 μg total protein).

• Gel selection: Use 10% SDS-PAGE gels to properly resolve the 45-50 kDa NR1I2 protein .

• Transfer conditions: Standard PVDF or nitrocellulose membranes are suitable.

• Blocking: Use 5% non-fat milk or BSA in TBS-T.

• Antibody dilution: Follow manufacturer recommendations, typically 1:500-1:2000 for primary antibody.

• Controls: Include positive control tissues known to express NR1I2 (liver, intestine).

• Expected molecular weight: Look for bands at 45-50 kDa, which is the observed molecular weight for NR1I2 .

What challenges might researchers face when studying NR1I2 methylation status?

Studying NR1I2 methylation presents several methodological challenges:

• CpG island location: The functionally significant CpG island is located around exon 3 of NR1I2, not in the conventional promoter region .

• Correlation with expression: Methylation status is inversely correlated with gene expression, as demonstrated in neuroblastoma studies .

• Tissue specificity: Methylation patterns may vary significantly between tissue types and disease states.

• Detection methods: Require specialized techniques such as bacterial artificial chromosome array-based methylated CpG island amplification (BAMCA) or bisulfite sequencing .

• Functional validation: Treatment with demethylating agents like 5-aza-2′-deoxycytidine can restore NR1I2 transcription in cell lines lacking endogenous expression, which should be used to confirm methylation-dependent silencing .

What are the recommended storage conditions for NR1I2 antibodies?

Most NR1I2 antibodies require specific storage conditions for optimal performance and longevity:

Always follow manufacturer-specific recommendations as formulations may vary between products .

How can I validate the specificity of my NR1I2 antibody?

To ensure antibody specificity:

• Perform knockdown/knockout experiments: Use siRNA or CRISPR to reduce NR1I2 expression and confirm corresponding reduction in antibody signal.

• Use multiple antibodies: Compare results with antibodies targeting different epitopes of NR1I2.

• Test reactivity across species: Confirm expected cross-reactivity matches the manufacturer's claims for human, mouse, rat, or other species .

• Check molecular weight: The observed molecular weight should be 45-50 kDa .

• Include appropriate controls: Use tissues/cells with known NR1I2 expression (e.g., liver) as positive controls.

• Peptide competition: If available, use the immunizing peptide to block specific binding.

• Verify with recombinant protein: Test antibody against purified recombinant NR1I2.

• Cross-application validation: Confirm consistent detection across multiple applications (WB, IHC, IF).

Why might I observe variable staining patterns with NR1I2 antibodies?

Variability in NR1I2 staining may result from:

• Isoform expression: Different tissues may express varying ratios of PXR1, PXR2, and other splice variants .

• Subcellular localization: NR1I2 shuttles between cytoplasm and nucleus depending on activation state.

• Epigenetic regulation: Methylation of NR1I2 can silence expression in certain tissues or disease states .

• Antibody epitope accessibility: Protein-protein interactions, particularly with RXR, may mask epitopes .

• Fixation and retrieval methods: Different protocols may affect epitope preservation.

• Activation state: Ligand binding can alter conformation and epitope exposure.

• Post-translational modifications: These may affect antibody recognition.

• Technical factors: Antibody concentration, incubation time, and detection methods influence staining intensity.

How can NR1I2 antibodies be used to study drug metabolism pathways?

NR1I2 antibodies enable several advanced approaches to drug metabolism research:

• Chromatin Immunoprecipitation (ChIP): Identify genomic binding sites of NR1I2 to map drug-responsive elements in target genes.

• Co-immunoprecipitation: Study NR1I2 interactions with RXR and other proteins in response to drug treatments .

• Protein expression profiling: Monitor NR1I2 expression across tissues to map drug metabolism potential.

• Subcellular trafficking: Track nuclear translocation upon drug exposure using immunofluorescence.

• Correlation studies: Examine relationships between NR1I2 levels and expression of drug-metabolizing enzymes.

• Drug-induced modifications: Investigate post-translational modifications of NR1I2 following drug exposure.

• Tissue-specific effects: Compare NR1I2 activation patterns across different organs in response to xenobiotics.

What role does NR1I2 play in cancer research and how can antibodies facilitate these studies?

NR1I2 has emerging significance in cancer research:

• Expression correlation: NR1I2 methylation and silencing has been observed in aggressive neuroblastomas, particularly those with MYCN amplification .

• Growth regulation: NR1I2 has demonstrated growth-suppressive activity in neuroblastoma cells .

• Biomarker potential: The prevalence of NR1I2 silencing in aggressive tumors suggests it could serve as a diagnostic marker to predict neuroblastoma prognosis .

• Target gene identification: Expression array analysis can reveal transcriptional targets of NR1I2 in cancer contexts .

• Therapeutic implications: Understanding NR1I2-regulated pathways may identify novel intervention points.

Antibodies enable these studies through expression profiling in tumors, subcellular localization analysis, and protein interaction studies.

How can researchers investigate NR1I2 in xenobiotic response mechanisms?

To study NR1I2's role in xenobiotic responses:

• Activation studies: Monitor nuclear translocation of NR1I2 after xenobiotic exposure using immunofluorescence.

• Target gene analysis: Combine ChIP with NR1I2 antibodies and expression studies to identify xenobiotic-responsive genes.

• Species comparisons: Use antibodies with cross-species reactivity to compare NR1I2 responses across human, mouse, and rat models .

• Tissue-specific patterns: Map expression and activation across metabolically relevant tissues (liver, intestine, kidney).

• Interaction profiling: Identify co-factors that modulate NR1I2 activity in response to different xenobiotics.

• Ligand specificity: Compare NR1I2 responses to various xenobiotics including drugs, environmental chemicals, and endogenous compounds.

• Regulatory pathways: Investigate upstream regulators and downstream effectors of NR1I2-mediated responses.

How should researchers interpret different molecular weights observed in Western blots for NR1I2?

When analyzing Western blot results:

• Expected molecular weight: The calculated molecular weight for NR1I2 is approximately 50-52 kDa, but the observed weight is typically 45-50 kDa .

• Higher molecular weight bands may indicate:

  • Post-translational modifications

  • Different isoforms (PXR2 at 473 aa vs. PXR1 at 434 aa)

  • Protein complexes not fully denatured

• Lower molecular weight bands may suggest:

  • Proteolytic degradation

  • Alternative splice variants

  • Truncated forms

• Multiple bands may represent:

  • A mixture of isoforms

  • Varied modification states

  • Partial degradation products

• Tissue-specific patterns may reflect differential expression of variants or processing.

Compare results to positive controls and recombinant standards when possible.

What are common technical pitfalls when using NR1I2 antibodies in various applications?

Researchers should be aware of these potential issues:

Additionally, be aware that antibody performance may vary across lots, and validation with appropriate controls is essential for all applications.

How do epigenetic modifications affect NR1I2 detection and function?

Epigenetic modifications significantly impact NR1I2 research:

• Methylation status: CpG island methylation around exon 3 of NR1I2 correlates inversely with gene expression .

• Detection challenges: Silenced NR1I2 due to methylation may result in false negatives in expression studies.

• Tissue/disease specificity: Methylation patterns vary across tissues and disease states, such as neuroblastoma with MYCN amplification .

• Functional consequences: Methylation-associated silencing affects NR1I2's growth-suppressive activities in certain cancers .

• Experimental approaches: Treatment with demethylating agents can restore expression, providing a tool to study epigenetic regulation .

• Promoter activity: The CpG island around exon 3 shows promoter activity, and its methylation status directly correlates with expression levels .

Researchers should consider these epigenetic influences when interpreting NR1I2 antibody results, particularly in cancer studies.