NR5A2 Antibody, HRP conjugated

What is NR5A2 and why is it significant in research?

NR5A2 (Nuclear receptor subfamily 5 group A member 2) is a DNA-binding zinc finger transcription factor belonging to the fushi tarazu factor-1 subfamily of orphan nuclear receptors. It functions as a key metabolic sensor by regulating genes involved in bile acid synthesis, cholesterol homeostasis, and triglyceride synthesis. NR5A2 plays crucial roles in liver metabolism, pancreatic function, inflammatory responses, and has been implicated in various disease states including metabolic syndrome and cancer progression . Its importance spans multiple research fields including metabolism, inflammation, and oncology.

What applications are NR5A2 antibodies typically used for?

NR5A2 antibodies are employed across multiple experimental applications:

• Western Blot (WB): Typically at dilutions of 1:1000-1:5000

• Immunohistochemistry (IHC-P): Usually at dilutions of 1:50-1:200

• Immunofluorescence/Immunocytochemistry (IF/ICC): Typically at dilutions of 1:50-1:200

• ELISA: Starting concentrations of approximately 1 μg/mL

• Immunoprecipitation (IP)

The specific dilution should be optimized for each assay system and antibody formulation .

What is the difference between HRP-conjugated and unconjugated NR5A2 antibodies?

HRP (Horseradish Peroxidase)-conjugated NR5A2 antibodies have the enzyme directly attached to the antibody molecule, eliminating the need for secondary antibody incubation in detection systems. This provides several advantages:

• Streamlined experimental workflows with fewer incubation steps

• Reduced background signal from secondary antibody cross-reactivity

• Enhanced sensitivity in certain applications, particularly IHC and WB

• Direct detection capabilities

Unconjugated antibodies require a secondary antibody conjugated to a detection system. The choice depends on your experimental design, with HRP-conjugated antibodies being particularly useful for direct detection methods .

How should I optimize NR5A2 antibody concentration for Western blot applications?

Optimizing NR5A2 antibody concentration for Western blot requires systematic titration:

• Begin with manufacturer's recommended dilution range (typically 1:1000-1:5000 for NR5A2)

• Perform a gradient dilution experiment (e.g., 1:500, 1:1000, 1:2000, 1:5000)

• Use appropriate positive controls (A549 cells, BxPC-3 cells, mouse liver tissue, or mouse pancreas tissue have shown reliable NR5A2 expression)

• Evaluate signal-to-noise ratio at each concentration

• Consider additional optimization parameters:

  • Blocking reagent composition (BSA vs. milk)

  • Incubation time and temperature

  • Washing stringency

For HRP-conjugated NR5A2 antibodies, a typical recommended dilution is 1:2000 for WB applications, but this should be optimized for your specific experimental system .

What are the critical parameters for successful immunohistochemistry with NR5A2 antibodies?

For optimal IHC results with NR5A2 antibodies:

• Tissue fixation and processing:

  • Use 10% neutral buffered formalin with controlled fixation time

  • Optimize antigen retrieval methods (heat-induced in citrate buffer pH 6.0 works well for NR5A2)

• Antibody parameters:

  • Start with manufacturer's recommended dilution (typically 1:150 for HRP-conjugated NR5A2 antibodies)

  • Extended incubation at 4°C overnight may improve specific binding

• Detection optimization:

  • For HRP-conjugated antibodies, ensure proper substrate exposure time

  • Use positive control tissues (liver, pancreas, testis, or ovary show reliable NR5A2 expression)

• Interpretation considerations:

  • NR5A2 shows primarily nuclear localization in most tissues

  • Expression intensity correlates with differentiation status in some cancers

Importantly, NR5A2 expression is higher in poorly differentiated tissues compared to well-differentiated keratinized strains in conditions like cSCC .

How can I use NR5A2 antibodies to investigate its role in metabolic syndrome and exercise physiology?

To investigate NR5A2's role in metabolic syndrome and exercise physiology:

• Experimental design considerations:

  • Use paired pre/post exercise samples from the same subjects

  • Include both acute and chronic exercise protocols

  • Compare samples from metabolic syndrome and healthy subjects

• Technical approach:

  • Quantify NR5A2 expression changes via Western blot or IHC

  • Co-stain with metabolic markers (glucose transporters, fatty acid metabolism enzymes)

  • Perform ChIP-seq to identify NR5A2 binding sites in metabolic genes

• Pathway analysis:

  • Focus on CYP7A1/CYP8B1 inflammatory pathway

  • Investigate C/EBPβ-FASN-SCD1 apoptotic pathway

  • Examine ALDH1B1 expression as downstream target

Research indicates that NR5A2 expression is downregulated in metabolic syndrome but can be upregulated through exercise, potentially through modulation of glucose metabolism pathways and reduction of inflammatory markers .

What approaches can be used to study NR5A2's role in inflammation and pyroptosis?

For investigating NR5A2's involvement in inflammation and pyroptosis:

• Cellular models:

  • Use Nr5a2 knockout cell lines (like LO2-NR5A2 variants)

  • Employ hepatocyte-specific Nr5a2 knockout mice models

• Key markers to examine:

  • Pyroptosis mediators: NLRP3, Caspase-1 (both pro- and cleaved forms), IL-1β

  • NF-κB pathway components

  • ROS markers: MDA levels, oxidative stress indicators

• Mechanistic investigations:

  • ROS measurement through fluorescent probes

  • ALDH1B1 expression analysis as NR5A2 target

  • NF-κB pathway inhibitor studies

• Technical approaches:

  • Immunoblotting for protein expression changes

  • Immunofluorescence for subcellular localization

  • ELISA for secreted inflammatory mediators (IL-1β)

  • MDA assays for oxidative stress

Recent research has revealed that inhibition of NR5A2 triggers pyroptosis, primarily mediated by activation of the NF-κB pathway, with ROS production playing a key intermediate role in this process .

How can ChIP assays be optimized to study NR5A2 transcriptional regulation?

For optimizing Chromatin Immunoprecipitation (ChIP) assays with NR5A2 antibodies:

• Antibody selection:

  • Validate antibody specificity by Western blot prior to ChIP

  • Consider using multiple antibodies targeting different epitopes

  • For HRP-conjugated antibodies, enzymatic activity may interfere with ChIP; use unconjugated variants

• Chromatin preparation:

  • Optimize crosslinking time (8-10 minutes with 1% formaldehyde works well for most transcription factors)

  • Sonication conditions must be carefully titrated to yield 200-500bp fragments

• Binding site identification:

  • Target consensus sequences 5'-CAAGG-3' or 5'-CCTTG-3' for NR5A2

  • Known binding sites include ALDH1B1 promoter regions:

    • Human: positions −1610 to −1595bp, −83 to −68bp, and −61 to −39bp

    • Mouse: positions −1961 to −1936bp, −1854 to −1834bp, and −630 to −612bp

• Controls and validation:

  • Use IgG negative controls

  • Include positive control regions (ALDH1B1 promoter or CYP7A)

  • Validate findings with luciferase reporter assays for functional confirmation

Research has demonstrated that NR5A2 directly binds to the ALDH1B1 promoter to regulate its expression, with mutation of the binding site from 5'-CAAGG-3' to 5'-CAACT-3' abolishing this interaction .

How can I address non-specific binding when using NR5A2 antibodies?

To minimize non-specific binding with NR5A2 antibodies:

• Antibody-specific approaches:

  • Titrate antibody concentration (particularly important for HRP-conjugated antibodies)

  • Pre-absorb antibody with recombinant protein if cross-reactivity is observed

  • Use alternative clone or host species if persistent issues occur

• Blocking optimization:

  • Test different blocking agents (5% BSA often works better than milk for phospho-epitopes)

  • Extend blocking time to 2 hours at room temperature

  • Add 0.1-0.3% Triton X-100 for membrane permeabilization in IF/ICC

• Washing modifications:

  • Increase washing stringency (higher salt concentration or mild detergents)

  • Extend wash times between antibody incubations

  • Use TBS-T instead of PBS-T for phospho-epitopes

• Signal verification:

  • Always include a negative control without primary antibody

  • Use knockout/knockdown samples as definitive specificity controls

  • Verify band size (NR5A2 typically appears at 61-67 kDa) .

How do I interpret seemingly contradictory results between different NR5A2 detection methods?

When encountering contradictory results between different detection methods:

• Technical considerations:

  • Different epitope accessibility across techniques (WB detects denatured protein; IHC/IF detect native conformation)

  • Buffer conditions affect antibody performance differently across methods

  • HRP-conjugated antibodies may perform differently from unconjugated versions in certain applications

• Biological variables:

  • NR5A2 has multiple isoforms that may be detected differentially

  • Post-translational modifications affect epitope recognition

  • Subcellular localization differences (primarily nuclear, but can vary with cellular state)

• Validation approaches:

  • Use multiple antibodies targeting different epitopes

  • Employ genetic knockdown/knockout controls

  • Correlate with mRNA expression data

  • Consider complementary techniques (mass spectrometry)

• Data interpretation:

  • Establish clear positivity criteria for each method

  • Document all experimental conditions thoroughly

  • Consider context-dependent expression patterns

NR5A2 expression patterns can vary significantly between tissues and disease states, with notable differences observed between normal and cancerous tissues .

How can NR5A2 antibodies be utilized to study its role in cancer progression?

For investigating NR5A2's involvement in cancer progression:

• Experimental models:

  • Patient-derived xenografts

  • NR5A2 knockdown/knockout cancer cell lines

  • Tissue microarrays comparing tumor grades

• Key relationships to examine:

  • NR5A2 expression correlation with tumor grade/stage

  • Association with Wnt/β-catenin signaling pathway components

  • Co-expression with other nuclear receptors

• Technical approaches:

  • Multiplex IHC for co-localization studies

  • RNA-seq paired with ChIP-seq for transcriptional networks

  • Tissue microarrays for high-throughput analysis

• Clinical correlations:

  • Compare expression across tumor grades and histological types

  • Correlate with patient outcomes and treatment response

  • Examine differences between primary and metastatic lesions

Recent research has revealed that NR5A2 expression is significantly higher in cutaneous squamous cell carcinoma (cSCC) tissues compared to healthy noncancerous tissues, with expression levels correlating with tumor grade. In cSCC, NR5A2 is primarily localized in the nucleus, and its expression is higher in poorly differentiated tissues compared to well-differentiated keratinized strains .

What considerations are important when using NR5A2 antibodies for studying its interactions with other transcription factors?

When investigating NR5A2 interactions with other transcription factors:

• Co-immunoprecipitation optimization:

  • Use mild lysis conditions to preserve protein-protein interactions

  • Consider crosslinking to stabilize transient interactions

  • Include appropriate controls (IgG, reverse IP)

  • For HRP-conjugated antibodies, enzymatic activity may interfere; use unconjugated alternatives

• Key interaction partners to examine:

  • AP-1 family members (c-Jun, JunB, JunD)

  • NR0B2 (co-repressor and target gene)

  • HNF1A (cooperates for hepatitis B virus gene transcription)

  • Oxysterol receptors NR1H3/LXR-alpha and NR1H2/LXR-beta

• Advanced techniques:

  • Proximity ligation assay for in situ interaction detection

  • Sequential ChIP for co-occupancy on shared target genes

  • FRET/BRET for real-time interaction monitoring

  • Mass spectrometry for unbiased interaction partner discovery

• Functional validation:

  • Luciferase reporter assays with wild-type and mutant binding sites

  • Gene expression analysis after partner knockdown

  • Mutation of interaction domains to disrupt specific partnerships

Research has demonstrated that NR5A2 undergoes a transcriptional switch in pancreatic tissue, relocating from differentiation-specific to inflammatory genes and promoting AP-1-dependent gene transcription. This process involves cooperation with c-Jun and is affected by the co-repressor NR0B2 .

What are the storage and handling recommendations for maintaining NR5A2 antibody activity?

For optimal maintenance of NR5A2 antibody activity:

• Storage conditions:

  • Store at -20°C as recommended by manufacturers

  • Avoid repeated freeze-thaw cycles (aliquot upon first thaw)

  • For HRP-conjugated antibodies, enzymatic activity is particularly sensitive to storage conditions

• Handling practices:

  • Centrifuge briefly before opening

  • Use sterile techniques when handling

  • Return to appropriate storage temperature immediately after use

• Stability considerations:

  • Most NR5A2 antibodies remain stable for 12 months from date of receipt

  • Working dilutions should be prepared fresh

  • Buffer composition affects stability (PBS with 0.05% proclin300, 50% glycerol, pH 7.3 is common)

• Quality control measures:

  • Periodically test antibody performance with positive controls

  • Document lot numbers and expiration dates

  • Consider stability testing if experiments span extended periods

Following manufacturer specifications is crucial, as different antibody formulations may have specific requirements for optimal preservation of both binding specificity and HRP enzymatic activity .

How do different detection systems compare when using HRP-conjugated NR5A2 antibodies?

When comparing detection systems for HRP-conjugated NR5A2 antibodies:

• Chromogenic detection systems:

  • DAB (3,3'-Diaminobenzidine): Produces brown precipitate, good stability

  • AEC (3-Amino-9-ethylcarbazole): Red precipitate, alcohol-soluble

  • TMB (3,3',5,5'-Tetramethylbenzidine): Blue precipitate, high sensitivity

• Chemiluminescent detection systems:

  • Enhanced chemiluminescence (ECL): Standard sensitivity

  • ECL Plus/Advanced: 5-10x more sensitive than standard ECL

  • Femto/SuperSignal: Highest sensitivity, useful for low abundance targets

• Factors affecting system selection:

  • Target abundance (NR5A2 expression varies by tissue type)

  • Required sensitivity and dynamic range

  • Image acquisition equipment availability

  • Need for multiplexing or quantification

• Optimization parameters:

  • Substrate exposure time (especially critical for chemiluminescence)

  • Substrate concentration and incubation temperature

  • Signal development time for chromogenic substrates

For NR5A2 detection in tissues with high expression (liver, pancreas), standard sensitivity systems are generally sufficient. For detection in tissues with lower expression or for quantitative analysis, enhanced sensitivity systems may be required .

What approaches are recommended for quantitative analysis of NR5A2 expression across different tissues?

For quantitative analysis of NR5A2 expression across tissues:

• Image analysis methods:

  • Use digital image analysis software with standardized protocols

  • Apply consistent thresholding for nuclear staining positivity

  • Consider H-score calculation: % positive cells × intensity (0-3)

  • For IF/IHC, measure both percentage positive cells and mean fluorescence/optical density

• Normalization strategies:

  • Use housekeeping proteins appropriate for tissue type

  • Consider tissue-specific expression benchmarks

  • Account for section thickness and fixation variables

  • Include calibration standards when comparing across experiments

• Statistical approaches:

  • Apply appropriate statistical tests for expression comparison

  • Use multiple reference tissues for relative expression calculation

  • Consider hierarchical clustering for expression pattern analysis

  • Account for biological variability with sufficient biological replicates

• Multi-omics integration:

  • Correlate protein expression with mRNA levels

  • Integrate with ChIP-seq data for functional relevance

  • Consider pathway analysis for contextual interpretation

Research has shown significant variations in NR5A2 expression across tissues, with high expression in liver, pancreas, intestine, and reproductive tissues. Expression patterns also vary in disease states, with increased expression in certain cancer types and decreased expression in metabolic syndrome .

How can NR5A2 binding site data be integrated with gene expression data to identify direct regulatory targets?

For integrating NR5A2 binding site data with gene expression:

• Experimental design:

  • Perform ChIP-seq for NR5A2 binding sites and RNA-seq for expression changes

  • Include both wild-type and NR5A2 knockdown/knockout conditions

  • Consider time-course experiments for dynamic regulation

• Bioinformatic analysis pipeline:

  • Identify NR5A2 binding motifs (consensus: 5'-CAAGG-3' or 5'-CCTTG-3')

  • Map binding sites to genomic features (promoters, enhancers)

  • Correlate binding intensity with expression changes

  • Perform Gene Ontology and pathway enrichment analysis

• Validation strategy:

  • Confirm direct binding with ChIP-qPCR for selected targets

  • Use luciferase reporter assays with wild-type and mutant binding sites

  • Perform site-directed mutagenesis of binding sites in model systems

  • Example validation cases include ALDH1B1 promoter regions:

    • Human: positions −1610 to −1595bp, −83 to −68bp, and −61 to −39bp

    • Mouse: positions −1961 to −1936bp, −1854 to −1834bp, and −630 to −612bp

• Integration with public datasets:

  • Cross-reference with public ChIP-seq datasets

  • Compare with expression databases across different conditions

  • Consider conservation analysis across species

Research has demonstrated that NR5A2 directly regulates genes involved in metabolism (CYP7A1/CYP8B1), inflammation, and cell differentiation through direct transcriptional regulation .

How should NR5A2 antibodies be used to investigate its role in nonalcoholic steatohepatitis (NASH)?

For investigating NR5A2's role in nonalcoholic steatohepatitis (NASH):

• Model systems selection:

  • High-fat diet-induced NASH mouse models

  • Hepatocyte-specific Nr5a2 knockout mice

  • Human NASH liver biopsies

• Technical approach:

  • IHC staining protocols optimized for fatty liver tissue

    • Extended deparaffinization steps

    • Modified antigen retrieval for lipid-rich samples

    • Careful background control (lipid deposits can cause non-specific binding)

  • Western blot considerations for lipid-rich tissues:

    • Modified extraction buffers to handle high lipid content

    • Additional centrifugation steps may be required

• Key markers to co-evaluate:

  • Pyroptosis markers: NLRP3, Caspase-1, IL-1β

  • Oxidative stress: ROS levels, MDA content

  • Fibrosis markers: α-SMA, collagen

  • Downstream targets: ALDH1B1, CYP7A1/CYP8B1

• Analytical considerations:

  • Zone-specific analysis in liver acinus

  • Correlation with disease severity and metabolic parameters

  • Consideration of treatment effects on NR5A2 expression

Recent research has shown that NR5A2 deficiency induces pyroptosis and promotes liver inflammation, suggesting a protective role in the development of NASH. The mechanism involves ROS-induced activation of the NF-κB pathway and downstream inflammatory mediators .

What considerations are important when studying the role of NR5A2 in pancreatic inflammation and cancer?

For studying NR5A2 in pancreatic inflammation and cancer:

• Model system selection:

  • Nr5a2 haploinsufficient mice

  • Caerulein-induced pancreatitis models

  • Patient-derived pancreatic cancer organoids

• Technical approach for pancreatic tissue:

  • Modified fixation protocols for pancreatic tissue (high protease content)

  • Rapid processing to prevent autodigestion

  • Careful interpretation of acinar vs. ductal components

• Key pathways and markers:

  • AP-1 signaling components (c-Jun, JunB, JunD)

  • Differentiation markers vs. inflammatory gene signatures

  • NR0B2 co-repressor expression and localization

• Experimental design considerations:

  • Time-course experiments to capture dynamic changes

  • Comparison between acute and chronic inflammation

  • Integration of genomic, transcriptomic, and proteomic approaches

Research has demonstrated that in Nr5a2 haploinsufficient mice, Nr5a2 undergoes a dramatic transcriptional switch, relocating from differentiation-specific to inflammatory genes and promoting AP-1-dependent gene transcription. This switch involves c-Jun and other inflammatory mediators and may contribute to both pancreatic inflammation and cancer development. Pancreatic deletion of c-Jun rescues the pre-inflammatory phenotype and the defective regenerative response to damage .

Human pancreata with reduced NR5A2 mRNA expression show histological changes reminiscent of early stages of pancreatitis-induced inflammation, suggesting that NR5A2 functions at the interface between differentiation and inflammation in the pancreas .

How might single-cell analysis techniques enhance our understanding of NR5A2 function?

Single-cell analysis techniques offer powerful new approaches to understand NR5A2 function:

• Single-cell RNA sequencing applications:

  • Identification of cell type-specific NR5A2 expression patterns

  • Discovery of heterogeneous responses to NR5A2 modulation

  • Characterization of rare cell populations with unique NR5A2 functions

• Single-cell protein analysis:

  • Mass cytometry (CyTOF) for multi-parameter protein expression

  • Single-cell Western blot for protein isoform analysis

  • Imaging mass cytometry for spatial context of NR5A2 expression

• Multi-omics integration at single-cell level:

  • CITE-seq for simultaneous RNA and protein measurement

  • Single-cell ATAC-seq for chromatin accessibility correlation

  • Spatial transcriptomics for tissue context of expression patterns

• Analytical considerations:

  • Trajectory analysis for developmental/differentiation processes

  • Regulatory network reconstruction at single-cell resolution

  • Identification of biomarker signatures for disease states

These approaches could reveal cell-specific functions of NR5A2 in heterogeneous tissues like liver and pancreas, potentially uncovering specialized roles in subpopulations that are masked in bulk tissue analysis. This is particularly relevant given NR5A2's known roles in both differentiation and inflammation pathways .

What are the prospects for developing NR5A2-targeted therapeutics based on current research?

Prospects for NR5A2-targeted therapeutics:

• Therapeutic rationales:

  • Metabolic disorders: NR5A2 activation may improve metabolic parameters

  • Inflammatory conditions: NR5A2 modulation could suppress inflammatory pathways

  • Cancer: Context-dependent targeting may be beneficial

• Current approaches in development:

  • Small molecule modulators of NR5A2 activity

  • Gene therapy approaches to restore NR5A2 expression

  • Combination therapies targeting NR5A2 and interacting pathways

• Challenges and considerations:

  • Tissue-specific effects of NR5A2 modulation

  • Context-dependent functions (pro- vs. anti-inflammatory)

  • Potential off-target effects on other nuclear receptors

  • Delivery methods to target specific tissues

• Biomarker development:

  • NR5A2 expression levels as predictive/prognostic markers

  • Downstream target expression patterns

  • Integration with existing clinical parameters