NFE2L3 Antibody, Biotin conjugated

What is NFE2L3 and what are its primary biological functions?

NFE2L3 (Nuclear Factor Erythroid 2-Related Factor 3), also known as NRF3, is a membrane-bound glycoprotein that is targeted to the endoplasmic reticulum and nuclear envelope . It functions as a transcription factor belonging to the cap 'n' collar (CNC) family. NFE2L3 has been identified as a negative upstream regulator of p53 and plays roles in various cellular processes . Recent research indicates NFE2L3 may have significant functions in regulating immune microenvironments, particularly in cancer contexts. In kidney cancer studies, NFE2L3 has been associated with immune-relevant pathways and correlates with the infiltration of multiple immune cell types, suggesting its potential role in modulating the tumor immune microenvironment .

What is a biotin-conjugated antibody and how does conjugation affect application?

A biotin-conjugated antibody is an immunoglobulin that has been chemically labeled with biotin molecules. This conjugation enhances the utility of the antibody in various detection methods due to biotin's strong affinity for avidin and streptavidin proteins. The NFE2L3 Antibody, Biotin conjugated (such as product A72130) is specifically designed with this modification to improve detection sensitivity in applications like ELISA .

The biotin conjugation offers several methodological advantages:

• Enhanced signal amplification through avidin-biotin interaction systems

• Improved detection sensitivity in complex biological samples

• Compatibility with multiple detection platforms

• Versatility in multi-color immunostaining protocols

The biotin-conjugated NFE2L3 antibody is typically supplied in liquid form with specific buffer components (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) that maintain stability and activity .

What are the recommended storage and handling conditions for NFE2L3 antibody?

For optimal preservation of NFE2L3 antibody activity, the following storage and handling protocols are recommended:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles that can degrade antibody quality

• For the biotin-conjugated version, aliquoting is generally unnecessary for -20°C storage

• When working with smaller quantities (20μl), solutions containing 0.1% BSA help maintain stability

• The storage buffer (typically PBS with 0.02% sodium azide and 50% glycerol at pH 7.3) should not be altered

Proper storage ensures antibody performance across applications and extends shelf life for ongoing research applications.

What applications are NFE2L3 antibodies validated for?

NFE2L3 antibodies have been validated for multiple research applications, with specific applications varying by product and conjugation type:

Published applications include knockdown/knockout validation studies, with at least three publications specifically utilizing Western blot techniques . The reactivity of these antibodies is predominantly with human samples, making them suitable for human-focused research .

How can NFE2L3 antibodies be utilized to investigate immune microenvironment regulation in cancer?

NFE2L3 has emerged as a potential functional gene regulating immune microenvironments in cancer, particularly in renal cell carcinoma. Methodological approaches for investigating this relationship include:

• Correlation analysis between NFE2L3 expression and immune cell infiltration:

  • NFE2L3 expression has been positively associated with infiltration of 17 types of immune cells, including naive CD4+ cells, CD8+ T cells, dendritic cells, and regulatory T cells

  • Specific techniques like ImmuCellAI and CIBERSORT can quantify these relationships

• Pathway analysis to identify immunomodulatory mechanisms:

  • Gene Set Enrichment Analysis (GSEA) reveals NFE2L3 association with immune-related processes, including adaptive immune response, T cell activation, and leukocyte cell-cell adhesion

  • NFE2L3 correlates with pathways involving NF-κB signaling, cytokine-cytokine receptor interaction, and NOD-like receptor signaling

• Methodological protocol:

  • Apply anti-NFE2L3 antibodies in immunohistochemistry to stratify tumors by NFE2L3 expression

  • Compare immune infiltration profiles between NFE2L3-high and NFE2L3-low groups

  • Correlate findings with clinical outcomes and mutation profiles

This approach has revealed that NFE2L3 expression correlates with immune signatures including immune checkpoint molecules, effector CD8 T cells, and antigen processing machinery in kidney cancer .

What are the optimal conditions for using NFE2L3 antibody in Western blot applications?

Optimizing Western blot protocols for NFE2L3 detection requires careful consideration of several parameters:

• Sample preparation:

  • NFE2L3 is a 76 kDa protein (694 amino acids)

  • Effective protein extraction requires buffers that can solubilize both membrane and nuclear proteins

• Antibody selection and dilution:

  • Unconjugated polyclonal antibodies (e.g., 17745-1-AP) are recommended at dilutions of 1:500-1:1000

  • Sample-dependent optimization may be necessary

• Blocking and washing conditions:

  • Standard blocking with 5% non-fat milk or BSA in TBST

  • Thorough washing steps to minimize background

• Detection considerations:

  • NFE2L3 detection may require enhanced chemiluminescence systems

  • Expected band size is approximately 76 kDa, though post-translational modifications may alter migration

• Validation approaches:

  • Positive controls include human placenta tissue, which has been validated for NFE2L3 expression

  • Knockdown/knockout validation can confirm antibody specificity

These parameters should be optimized based on specific experimental contexts and sample types.

How can researchers validate the specificity of NFE2L3 antibodies in their experimental systems?

Validating antibody specificity is crucial for generating reliable research data. For NFE2L3 antibodies, several validation approaches are recommended:

• Genetic validation:

  • Knockdown/knockout experiments using siRNA or CRISPR-Cas9

  • Multiple published studies have employed this approach for NFE2L3 antibody validation

• Peptide competition assays:

  • Using the immunogenic peptide (e.g., amino acids 132-260 or 666-694 for different antibodies)

  • Gradual reduction in signal intensity confirms specificity

• Cross-platform validation:

  • Comparing results across multiple detection methods (WB, IF, ICC, ELISA)

  • Consistency across techniques supports specificity

• Positive and negative control tissues:

  • Human placenta tissue serves as a positive control

  • Tissue known to lack NFE2L3 expression can serve as negative controls

• Molecular weight confirmation:

  • The observed molecular weight (76 kDa) should match the predicted size

  • Any deviations might indicate post-translational modifications or splice variants

What considerations are important when using NFE2L3 antibodies for prognostic biomarker development?

NFE2L3 has demonstrated potential as a prognostic biomarker, particularly in renal cancer. Researchers developing NFE2L3-based prognostic tools should consider:

• Expression pattern analysis:

  • NFE2L3 shows elevated expression in kidney renal clear cell carcinoma (KIRC) and kidney renal papillary cell carcinoma (KIRP) compared to adjacent normal tissues

  • This differential expression correlates with poor patient outcomes

• Antibody selection criteria:

  • Epitope selection is crucial (various antibodies target different regions: AA 132-260, 300-550, 351-400, 666-694)

  • Consider antibody format based on intended application (unconjugated for IHC/WB, biotin-conjugated for sensitive detection systems)

• Standardization of protocols:

  • Establish robust cutoff values for "high" versus "low" expression

  • Standardize staining and scoring methodologies across patient cohorts

• Integration with other biomarkers:

  • NFE2L3 expression correlates with specific mutations (TP53, MET)

  • Multiparameter analysis may enhance prognostic value

• Validation across patient populations:

  • Independent cohort validation is essential

  • Consider demographic and disease subtype variations

The prognostic value of NFE2L3 should be evaluated in the context of existing clinical parameters to determine its incremental value in patient stratification.

What are the technical challenges in immunofluorescence applications with NFE2L3 antibodies?

Immunofluorescence (IF) applications with NFE2L3 antibodies present several technical challenges that researchers should address:

• Subcellular localization complexity:

  • NFE2L3 localizes to both membrane (endoplasmic reticulum) and nuclear compartments

  • Fixation methods may affect epitope accessibility differently in these compartments

• Optimization of antibody concentrations:

  • Recommended dilutions range from 1:50-1:500 for IF applications

  • Cell type-specific optimization is necessary (positive results have been documented in HepG2 cells)

• Signal amplification considerations:

  • Direct detection may be insufficient for low-abundance expression

  • Biotin-conjugated antibodies can be used with streptavidin-fluorophore systems

  • Tyramide signal amplification may enhance detection sensitivity

• Autofluorescence mitigation:

  • Tissue-specific autofluorescence can obscure specific signals

  • Quenching protocols may be necessary, especially in kidney tissue

• Co-localization studies:

  • Dual staining with organelle markers can confirm subcellular localization

  • Spectral unmixing may be necessary for multi-label experiments

Addressing these challenges requires systematic optimization and appropriate controls to ensure reliable and reproducible results.

How should researchers design experiments to investigate NFE2L3's role in cellular pathways?

Designing robust experiments to elucidate NFE2L3's role in cellular pathways requires careful consideration of multiple factors:

• Experimental model selection:

  • Cell lines with confirmed NFE2L3 expression (e.g., HepG2)

  • Primary human samples where appropriate

  • Consider cancer vs. normal tissue contexts based on research questions

• Pathway analysis approach:

  • Gene Set Enrichment Analysis (GSEA) has successfully identified NFE2L3-associated pathways

  • Focus on immune-related processes, NF-κB signaling, cytokine interactions, and NOD-like receptor signaling

• Functional validation methods:

  • Gene silencing (siRNA, shRNA) or overexpression

  • Antibody-based detection of downstream effectors

  • Pathway inhibitors to confirm mechanistic relationships

• Multidimensional data integration:

  • Correlate protein expression with transcriptomic data

  • Analyze co-expressed genes (e.g., the 2310 NFE2L3 co-expressed genes identified in previous studies)

  • Connect findings to functional outcomes (proliferation, migration, immune response)

• Temporal considerations:

  • Acute vs. chronic effects of NFE2L3 modulation

  • Cell cycle-dependent variations in pathway activity

This methodological framework enables systematic investigation of NFE2L3's regulatory roles in complex cellular networks.

What are common troubleshooting issues when using NFE2L3 antibodies and how can they be resolved?

Researchers frequently encounter technical challenges when working with NFE2L3 antibodies. Here are common issues and their solutions:

• Issue: High background in Western blots
  Solution:

  • Increase blocking stringency (5% BSA instead of milk)

  • Extend blocking time to 2 hours at room temperature

  • Optimize antibody dilution (start with 1:1000)

  • Increase washing duration and frequency

• Issue: No signal detection
  Solution:

  • Confirm NFE2L3 expression in your sample (human placenta as positive control)

  • Verify protein transfer efficiency

  • Try alternative epitope antibodies (various regions: AA 132-260, 300-550, 351-400, 666-694)

  • Employ signal enhancement techniques (biotin-streptavidin systems)

• Issue: Multiple bands in Western blot
  Solution:

  • Validate with knockout/knockdown controls

  • Consider post-translational modifications or splice variants

  • Optimize SDS-PAGE conditions (gradient gels may help resolve closely migrating bands)

  • Use more specific antibodies targeting defined epitopes

• Issue: Weak or inconsistent immunofluorescence signals
  Solution:

  • Optimize fixation protocols (paraformaldehyde vs. methanol)

  • Test permeabilization conditions for nuclear epitope access

  • Implement antigen retrieval methods

  • Increase antibody concentration within recommended range (1:50-1:500)

• Issue: Biotin-conjugated antibody exhibits non-specific binding
  Solution:

  • Block endogenous biotin with avidin/biotin blocking kit

  • Optimize detection reagent concentration

  • Increase washing stringency

  • Consider alternative conjugates if problems persist

These troubleshooting approaches should be systematically implemented to resolve technical challenges.

How does the selection of NFE2L3 antibody epitope affect experimental outcomes?

The choice of epitope target in NFE2L3 antibodies significantly impacts experimental outcomes across different applications:

• Epitope location considerations:

  • N-terminal epitopes (AA 132-260) may detect different isoforms than C-terminal epitopes (AA 666-694)

  • Internal regions (AA 300-550, 351-400) may offer differential access in certain applications

  • Some epitopes may be masked by protein-protein interactions or post-translational modifications

• Application-specific implications:

  • For Western blot applications, epitope accessibility after denaturation is critical

  • For immunofluorescence, native protein conformation may render certain epitopes inaccessible

  • For ELISA, surface-exposed epitopes in solution provide optimal detection

• Correlating epitope selection with functional domains:

  • Regulatory domains may be masked in certain cellular contexts

  • DNA-binding domains might be accessible only in specific activation states

  • Membrane-associated regions may require special extraction protocols

• Experimental validation approach:

  • Compare multiple antibodies targeting different epitopes

  • Correlate findings with functional outcomes

  • Document epitope-specific differences in published results

• Technical implications of epitope selection:

  • Purification methods vary (protein A vs. peptide affinity purification)

  • Recognition of post-translationally modified forms may vary by epitope

  • Cross-reactivity profiles differ between epitope regions

Researchers should select epitopes based on their specific experimental questions and validation requirements.

How can NFE2L3 antibodies be used to investigate its role in cancer immune evasion mechanisms?

The emerging role of NFE2L3 in immune regulation suggests potential involvement in cancer immune evasion. Researchers can investigate this connection using the following methodological approaches:

• Correlation analysis with immune checkpoint molecules:

  • NFE2L3 expression has been correlated with immune checkpoints in kidney cancer

  • Use anti-NFE2L3 antibodies to stratify tumors, then analyze checkpoint molecule expression

• Mechanistic studies of NFE2L3-mediated immune modulation:

  • Modulate NFE2L3 expression in cancer cell lines

  • Co-culture with immune cells and assess functional outcomes

  • Measure cytokine profiles and immune cell activation markers

• Analysis of NFE2L3 in the tumor microenvironment:

  • Multiplex immunofluorescence with NFE2L3 and immune cell markers

  • Spatial relationship analysis between NFE2L3-expressing cells and immune infiltrates

  • Correlation with clinical outcomes and treatment response

• Integration with mutation data:

  • NFE2L3 expression changes correlate with specific mutations (TP53, MET)

  • Analyze how these genetic alterations affect immune evasion mechanisms

• Therapeutic targeting potential:

  • Assess how NFE2L3 modulation affects response to immunotherapy

  • Investigate combination approaches targeting NFE2L3 and immune checkpoints

This research direction could yield insights into novel immunotherapeutic approaches for cancers with NFE2L3 dysregulation.

What methodological approaches can resolve contradictory findings in NFE2L3 research?

Research on NFE2L3 function has sometimes produced seemingly contradictory results. Resolving these contradictions requires systematic methodological approaches:

• Cell type and context specificity analysis:

  • NFE2L3 functions may differ between cell types

  • Compare findings across multiple cell lines and primary tissues

  • Document specific experimental conditions that produce different outcomes

• Isoform-specific investigation:

  • Use epitope-specific antibodies to distinguish NFE2L3 isoforms

  • Perform isoform-specific knockdown/overexpression experiments

  • Correlate isoform expression with functional outcomes

• Post-translational modification characterization:

  • Investigate phosphorylation, glycosylation, or other modifications

  • Use modification-specific antibodies or mass spectrometry

  • Correlate modifications with subcellular localization and function

• Temporal dynamics consideration:

  • Acute vs. chronic effects may differ

  • Design time-course experiments with appropriate controls

  • Document cell cycle-dependent variations

• Technical validation and standardization:

  • Cross-validate findings using multiple antibodies and techniques

  • Standardize experimental protocols across research groups

  • Establish positive and negative controls for each experimental system

Systematic application of these approaches can help reconcile apparently contradictory findings and build a more coherent understanding of NFE2L3 biology.

How can multiplexing approaches be optimized when working with NFE2L3 antibodies?

Multiplexing technologies enable simultaneous analysis of NFE2L3 alongside other markers, providing richer contextual data. Optimizing these approaches requires:

• Antibody compatibility assessment:

  • Test for cross-reactivity between antibodies in the panel

  • Ensure spectral separation of fluorophores

  • Validate each antibody individually before multiplexing

• Sequential immunostaining protocol development:

  • Optimize order of antibody application (typically start with lowest abundance target)

  • Include stripping/blocking steps between rounds

  • Validate signal preservation during multi-round protocols

• Multiplexed immunofluorescence optimization:

  • For biotin-conjugated NFE2L3 antibodies, employ orthogonal detection systems for other targets

  • Use spectrally distinct fluorophores with minimal overlap

  • Implement computational unmixing for closely spaced emission spectra

• Mass cytometry/CyTOF adaptation:

  • Metal-conjugated NFE2L3 antibodies enable high-parameter analysis

  • Validate metal-conjugated antibodies against traditional fluorescent versions

  • Optimize staining conditions for complex cell suspensions

• Spatial profiling considerations:

  • In situ hybridization combined with NFE2L3 immunodetection

  • Digital spatial profiling with region selection based on NFE2L3 expression

  • Correlation of spatial data with functional outcomes

These methodological refinements enable complex analyses of NFE2L3 in relation to other markers and cellular contexts.

What are the considerations for using NFE2L3 antibodies in translational research and potential clinical applications?

Translational applications of NFE2L3 research, particularly its potential as a biomarker, require specific methodological considerations:

• Analytical validation requirements:

  • Establish reproducibility across laboratories

  • Determine analytical sensitivity and specificity

  • Define ranges for normal versus pathological expression

• Clinical sample handling optimization:

  • Standardize fixation protocols for FFPE tissues

  • Establish optimal antigen retrieval methods

  • Validate across diverse patient populations

• Scoring system development:

  • Create quantitative assessment methods (H-score, Allred score)

  • Train multiple pathologists to ensure inter-observer reliability

  • Correlate scores with clinical outcomes

• Companion diagnostic potential:

  • Given NFE2L3's association with immune regulation , investigate its utility as a companion diagnostic for immunotherapies

  • Develop standardized assays with defined cutoffs

  • Validate in prospective clinical trials

• Regulatory considerations:

  • Document antibody validation according to CLIA/CAP guidelines

  • Establish standard operating procedures for clinical laboratory implementation

  • Address quality control requirements for clinical application

These translational approaches can bridge the gap between basic NFE2L3 research and potential clinical applications, particularly in oncology.

What resources are available for NFE2L3 antibody validation and protocol optimization?

Researchers seeking to optimize their NFE2L3 antibody protocols can access several resources:

• Published validation studies:

  • At least three published works have utilized NFE2L3 antibodies in Western blot applications

  • One study demonstrating NFE2L3's role in kidney cancer immune regulation provides detailed methodological information

• Manufacturer technical resources:

  • Detailed product information sheets specifying recommended dilutions (WB: 1:500-1:1000, IF/ICC: 1:50-1:500)

  • Validation galleries showing expected results in specific applications

  • Technical support services for troubleshooting

• Online protocol repositories:

  • Protocols optimized for different applications (Western blot, immunofluorescence, ELISA)

  • Application notes for specific cell types and tissues

• Reference materials:

  • Positive control samples (human placenta tissue)

  • Recombinant proteins for antibody validation

• Database resources:

  • UniProt entry (Q9Y4A8) providing sequence and domain information

  • GenBank accession (BC068455) for reference sequence verification

These resources provide valuable starting points for optimizing NFE2L3 antibody applications in various research contexts.

How can researchers effectively compare different NFE2L3 antibody products for their specific applications?

When selecting an NFE2L3 antibody for specific research applications, systematic comparison is essential:

• Epitope comparison framework:

  • Map available antibodies to specific regions (AA 132-260, 300-550, 351-400, 666-694)

  • Consider epitope conservation across species if working with non-human models

  • Evaluate epitope accessibility in your experimental system

• Application-specific performance metrics:

  • For Western blot: sensitivity, specificity, background levels

  • For immunofluorescence: signal-to-noise ratio, subcellular localization precision

  • For ELISA: detection limit, dynamic range, reproducibility

• Conjugation options assessment:

  • Unconjugated for maximum flexibility

  • Biotin-conjugated for signal amplification systems

  • Other conjugates (FITC, HRP) for direct detection

• Validation evidence evaluation:

  • Published literature using specific antibody clones

  • Manufacturer validation data (knockdown/knockout controls)

  • Independent validation by research community

• Technical specifications comparison:

  • Host species and clonality (all reported NFE2L3 antibodies are rabbit polyclonal)

  • Purification method (protein A, protein G, peptide affinity)

  • Buffer composition and storage requirements

This systematic comparison approach enables informed selection of the most appropriate NFE2L3 antibody for specific research questions.