NFE2L2 Antibody

What is the molecular weight pattern expected when detecting NFE2L2/NRF2 by Western blot?

NFE2L2/NRF2 typically displays multiple banding patterns in Western blot experiments, which can cause confusion among researchers. The predicted molecular weight is approximately 68 kDa, but the observed molecular weight often appears at both 68 kDa and 95-110 kDa due to post-translational modifications . According to validation studies, the consensus in scientific literature confirms that the actual observed molecular weights of NFE2L2 are approximately 70 kDa and 95-110 kDa . This variation in banding pattern can lead to uncertainties about specificity, which requires careful validation of antibodies used.

What are the recommended dilutions for NFE2L2 antibodies across different applications?

Different experimental applications require specific antibody dilution ranges for optimal results:

It's important to note that these dilutions should be optimized for each specific experimental system, as reactivity can vary depending on sample type and processing methods . For recombinant monoclonal antibodies, manufacturers may recommend different dilutions, such as 1:20-1:200 for IF applications .

How can I improve detection of endogenous NFE2L2 protein under basal conditions?

Under normal physiological conditions, NFE2L2 is rapidly ubiquitinated and degraded in the cytoplasm, making it difficult to detect by Western blot . Methodological approaches to improve detection include:

• Adding proteasome inhibitors like MG132 to cell cultures prior to lysis, which effectively inhibits NFE2L2 degradation and increases detection signal

• Using nuclear extraction protocols since NFE2L2 accumulates in the nucleus during oxidative stress

• Employing treatments that induce oxidative stress (e.g., H₂O₂) or chemical activators like sulforaphane (SFN) to stabilize NFE2L2

• Optimizing lysis buffers to include phosphatase inhibitors that preserve post-translational modifications

These approaches can significantly enhance the detection of endogenous NFE2L2 in experimental systems .

What cell types and tissues are recommended for validating NFE2L2 antibody specificity?

Based on extensive validation studies, specific cell types and tissues have been consistently used to verify NFE2L2 antibody specificity:

These specific sample types have demonstrated reliable NFE2L2 detection and should be considered as positive controls when validating new antibodies or experimental conditions . Notably, antibodies may show differential reactivity across species, with most validated for human samples .

How can I design proper controls when studying NFE2L2 translocation in response to stress conditions?

Designing appropriate controls for NFE2L2 translocation studies requires multiple approaches:

• Positive controls: Include known NFE2L2 activators such as sulforaphane (SFN) treatment, which has been demonstrated to induce NFE2L2 nuclear accumulation and target gene expression

• Negative controls: Use Nfe2l2-knockout cells or tissues as definitive negative controls, which have been shown to exhibit reduced expression of autophagy genes including Sqstm1, Calcoco2, Ulk1, Atg2b, Atg4d, Atg5, Atg7, and Gabarapl1

• Time-course experiments: Monitor NFE2L2 translocation at multiple time points following stress induction (e.g., 0, 1, 2, 4, and 8 hours post-treatment)

• Subcellular fractionation: Compare cytoplasmic versus nuclear fractions to quantify translocation efficiency

• Rescue experiments: Transfection of NFE2L2 expression constructs (particularly those lacking the KEAP1 regulatory ETGE domain) into knockout cells can restore target gene expression and serve as functional validation

These control strategies ensure reliable interpretation of NFE2L2 translocation and downstream effects in response to various stimuli.

What are validated approaches for ChIP experiments targeting NFE2L2 given the challenges with endogenous immunoprecipitation?

ChIP experiments with NFE2L2 present unique challenges due to difficulties in immunoprecipitating endogenous NFE2L2 efficiently. Published protocols have successfully addressed this limitation through the following approaches:

• Using HEK293T cells transfected with an expression vector for V5-tagged NFE2L2 that lacks the KEAP1 regulatory domain (ETGE), facilitating NFE2L2 stabilization, nuclear translocation, and binding to target genes

• Performing ChIP with anti-V5 antibody (for tagged NFE2L2) and anti-IgG as negative control

• Analyzing immunoprecipitated DNA by quantitative real-time PCR (qRT-PCR) with specific primers surrounding putative Antioxidant Response Elements (AREs)

• Screening chromatin immunoprecipitation databases like ENCODE for proteins that bind NFE2L2-regulated enhancer AREs (such as MAFK and BACH1) as alternative approaches to identify NFE2L2 binding sites

• Validating newly identified ARE sequences in autophagy genes through enrichment analysis in V5-immunoprecipitated chromatin

These approaches have successfully identified NFE2L2 binding to regulatory regions of multiple autophagy genes, establishing its role in transcriptional regulation beyond oxidative stress response.

How can NFE2L2 antibodies be used to investigate its role in cancer and therapeutic resistance?

NFE2L2 antibodies serve as critical tools for investigating its role in cancer development and treatment resistance:

• Mutation analysis: NFE2L2 mutations, particularly in the ETGE or DLG domains that abrogate KEAP1 association, can be detected through immunoblotting to assess nuclear accumulation patterns

• Prognostic biomarker assessment: Studies have shown that NFE2L2/KEAP1/CUL3 mutations are associated with significantly more local failure (HR = 3.50, 95% CI: 1.56–7.89, P = 0.0025) and locoregional failure (HR = 3.80, 95% CI: 1.80–8.03, P = 0.0005) in cancer radiotherapy

• Therapeutic resistance monitoring: NFE2L2 nuclear accumulation can be quantified by immunohistochemistry or immunofluorescence to correlate with radiotherapy outcomes, as demonstrated in T2N0 glottic squamous cell carcinoma patients where disease-free survival was significantly worse for patients with NFE2L2 pathway mutations (HR = 2.88, 95% CI: 1.46–5.66, P = 0.0022)

• Oncogenic driver analysis: Patient-derived NFE2L2 mutants (e.g., L30P and R34P) have been shown to dramatically accelerate tumorigenesis when co-expressed with other oncogenic factors, which can be tracked using specific antibodies

These applications highlight NFE2L2 antibodies as valuable tools for understanding cancer biology and therapeutic resistance mechanisms.

What strategies can address inconsistent or background issues when using NFE2L2 antibodies in immunohistochemistry?

Researchers encountering inconsistent results or background issues with NFE2L2 antibodies in IHC can implement several validated strategies:

• Optimized antigen retrieval: For NFE2L2 detection in tissues, suggested protocols include antigen retrieval with TE buffer pH 9.0, although citrate buffer pH 6.0 may be used as an alternative

• Blocking optimization: Extended blocking periods (1-2 hours) with specialized blocking buffers containing both serum proteins and detergents can reduce nonspecific binding

• Antibody validation: Using tissues from nfe2l2-knockout mice as negative controls and tissues with known high NFE2L2 expression (e.g., human liver cancer tissue, human breast cancer tissue) as positive controls

• Secondary antibody selection: Higher dilutions of secondary antibodies (1:1000 or greater) often reduce nonspecific staining while maintaining specific signal

• Signal amplification systems: For low-expression samples, employing tyramide signal amplification systems can enhance specific signals while maintaining signal-to-noise ratio

These methodological refinements have been successful in multiple published studies and can significantly improve NFE2L2 detection specificity in tissue samples.

What are the best approaches for validating NFE2L2 antibody specificity for research publications?

Comprehensive validation of NFE2L2 antibodies for publication-quality research should include:

• Genetic controls: Testing antibodies on samples from Nfe2l2-knockout mice or cells with CRISPR/Cas9-mediated NFE2L2 deletion to demonstrate specificity

• Rescue experiments: Reintroducing NFE2L2 expression in knockout systems using constructs like NFE2L2-ΔETGE-V5, which has been shown to rescue normal basal levels of expression of autophagy genes including Sqstm1, Calcoco2, Ulk1, Atg5, Atg7, and Gabarapl1

• Multi-technique validation: Confirming consistent results across complementary techniques (e.g., if Western blot shows a 68 kDa band, validate with mass spectrometry or immunoprecipitation followed by protein identification)

• Known inducer testing: Demonstrating appropriate response to established NFE2L2 activators like sulforaphane or oxidative stress inducers (H₂O₂), which should show increased nuclear localization and target gene expression

• Cross-antibody validation: Using multiple antibodies targeting different epitopes of NFE2L2 to confirm consistent detection patterns

These validation approaches provide compelling evidence of antibody specificity and reliability, essential for high-quality publications.

How can NFE2L2 antibodies be used to investigate the crosstalk between oxidative stress response and autophagy?

Recent research has established NFE2L2 as a critical regulator of both oxidative stress response and autophagy. To investigate this crosstalk:

• Chromatin immunoprecipitation: NFE2L2 antibodies can be used in ChIP assays to identify direct binding to antioxidant response elements (AREs) in autophagy gene promoters. Studies have validated 11 ARE regions in autophagy genes that NFE2L2 directly binds, including SQSTM1, CALCOCO2, ULK1, ATG2B, ATG4D, ATG5, ATG7, and GABARAPL1

• Dual immunofluorescence: Co-staining of NFE2L2 with autophagy markers like SQSTM1/p62 can reveal their spatial relationship during stress conditions. Studies in Alzheimer's disease models show reduced colocalization of APP and MAPT with SQSTM1/p62 in the absence of NFE2L2, suggesting impaired autophagy-mediated clearance

• Autophagy flux assessment: NFE2L2 antibodies combined with LC3B antibodies can help assess autophagy flux under oxidative stress. Research shows that H₂O₂ treatment increases LC3B-II levels in wild-type cells but to a lesser extent in nfe2l2-KO MEFs, indicating NFE2L2 regulates autophagy specifically under oxidative stress conditions

• Rescue experiments: Introducing NFE2L2 constructs lacking the KEAP1 regulatory domain into NFE2L2-deficient cells restores expression of autophagy genes, providing a powerful tool to confirm direct regulation

These approaches have revealed that NFE2L2 functions at the intersection of redox homeostasis and protein quality control through direct transcriptional regulation of autophagy genes.

What are the current challenges and solutions for detecting modified forms of NFE2L2 protein?

NFE2L2 undergoes multiple post-translational modifications that affect its stability, localization, and function. Current challenges and solutions include:

• Phosphorylation detection: NFE2L2 is phosphorylated by multiple kinases, affecting its stability and activity. Phospho-specific antibodies combined with phosphatase inhibitors in lysis buffers can help detect these modified forms

• Ubiquitination analysis: Under normal conditions, NFE2L2 is heavily ubiquitinated. Using proteasome inhibitors (MG132) combined with immunoprecipitation and ubiquitin-specific antibodies allows detection of ubiquitinated forms

• Acetylation detection: NFE2L2 acetylation affects DNA binding. Immunoprecipitation with NFE2L2 antibodies followed by detection with acetyl-lysine antibodies can reveal this modification

• Nuclear vs. cytoplasmic forms: Different modified forms predominate in different cellular compartments. Subcellular fractionation combined with Western blotting can distinguish these populations

• High molecular weight bands: NFE2L2 often appears at 95-110 kDa in addition to the predicted 68 kDa band due to modifications . Using gradient gels (4-15%) improves resolution of these forms

Understanding these modified forms is critical, as they represent different functional states of NFE2L2 and may serve as more specific biomarkers in disease contexts.

How can NFE2L2 antibodies contribute to understanding neurodegenerative disease mechanisms?

NFE2L2 antibodies are proving valuable for investigating neurodegenerative disease mechanisms:

• Alzheimer's disease models: In mouse models co-expressing human APP V717I and MAPT P301L, NFE2L2 deficiency leads to increased intraneuronal protein aggregates and reduced levels of autophagy proteins (SQSTM1/p62, CALCOCO2/NDP52, ULK1, ATG5, GABARAPL1). NFE2L2 antibodies allow visualization of these changes through immunohistochemistry and immunofluorescence

• Human patient samples: In AD patients, neurons expressing high levels of APP or MAPT also express SQSTM1/p62 and nuclear NFE2L2, suggesting attempted clearance of intraneuronal aggregates through autophagy. This can be detected using co-immunostaining approaches

• Therapeutic response markers: As NFE2L2 activators emerge as potential therapeutics for neurodegenerative diseases, antibodies can be used to confirm target engagement and downstream pathway activation

• Selective vulnerability assessment: NFE2L2 antibodies can help identify neuronal populations with differential NFE2L2 activity, potentially explaining selective vulnerability in neurodegenerative diseases

These applications highlight NFE2L2 antibodies as important tools for both basic and translational research in neurodegenerative disease mechanisms.

What methodological considerations are important when using NFE2L2 antibodies to evaluate cancer therapeutic resistance mechanisms?

When studying cancer therapeutic resistance using NFE2L2 antibodies, several methodological considerations are crucial:

• Mutation-specific detection: NFE2L2 mutations in the ETGE or DLG domains that abrogate KEAP1 association lead to constitutive NFE2L2 nuclear translocation and therapeutic resistance. Antibodies must be validated to detect both wild-type and mutant forms equally

• Quantitative image analysis: For accurate assessment of nuclear/cytoplasmic ratios in patient samples, standardized immunohistochemistry protocols combined with digital image analysis are recommended

• Combination with genomic analysis: Recent clinical studies demonstrate that mutations in NFE2L2/KEAP1/CUL3 predict radiation treatment failure in T2N0 glottic cancer (HR = 3.50; 95% CI, 1.56–7.89; P = 0.0025 for local failure). Correlating antibody staining patterns with mutation status provides the most comprehensive assessment

• Temporal dynamics monitoring: Serial sampling during treatment can reveal evolution of NFE2L2 activation as resistance emerges

• Downstream target assessment: Combining NFE2L2 antibodies with antibodies against downstream targets provides functional validation of pathway activation

These methodological approaches support more precise characterization of NFE2L2's role in treatment resistance, potentially guiding personalized therapy decisions.