NFE2 Antibody

What is NFE2 and what biological functions does it regulate?

NFE2 (Nuclear Factor Erythroid 2) is a transcription factor essential for regulating erythroid and megakaryocytic maturation and differentiation. It functions as a component of the NF-E2 complex that binds to the hypersensitive site 2 (HS2) of the beta-globin control region (LCR). NFE2 recognizes the TCAT/C sequence of the AP-1-like core palindrome present in erythroid and megakaryocytic gene promoters . It plays a pivotal role in erythroid maturation and, together with other transcription factors such as EKLF and GATA1, controls the transcription of erythroid-specific genes, including β-globin . Importantly, NFE2 regulates multiple enzymes in the heme biosynthesis pathway, demonstrating its critical role in coordinating hemoglobin production .

How do I select the appropriate NFE2 antibody for my research application?

When selecting an NFE2 antibody, consider these methodological steps:

• Determine your experimental application requirements (WB, IHC, IF, IP, ChIP)

• Verify species reactivity (human, mouse, rat) matches your samples

• Check antibody validation data for your specific application

• Review published literature using the antibody for similar applications

• Consider antibody format (polyclonal vs monoclonal) based on research needs

For example, the polyclonal antibody 11089-1-AP has been validated for Western Blot (1:500-1:1000), Immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg lysate), IHC (1:20-1:200), and IF/ICC (1:20-1:200) with human samples . Always check antibody specificity by examining Western blot images showing the expected molecular weight (approximately 45 kDa for NFE2) .

What sample preparation methods are recommended for NFE2 detection?

For optimal NFE2 detection:

• Tissue samples for IHC: Perform heat-mediated antigen retrieval with citrate buffer pH 6.0 or TE buffer pH 9.0 before staining

• Cell lysates for Western blot: Extract proteins under conditions that preserve phosphorylation and other post-translational modifications

• Immunoprecipitation: Incubate antibody (e.g., 50 μg) with prepared homogenates for one hour at 4°C, followed by incubation with magnetic Protein A/G beads

• Chromatin immunoprecipitation: Use protocols optimized for transcription factor binding site analysis, as demonstrated in K562 cells for detecting NFE2 binding to heme biosynthesis enzyme promoters

How can I determine if my NFE2 antibody is detecting post-translationally modified forms of the protein?

Post-translational modifications (PTMs) significantly affect NFE2 function. To detect PTM-specific forms:

• Use phosphatase treatment on parallel samples to identify phosphorylated NFE2

• Compare reducing and non-reducing conditions in Western blot

• Utilize size-separation techniques to differentiate between modified forms (NFE2 can appear at ~45 kDa (unmodified) or higher molecular weights)

• Consider immunoprecipitation followed by mass spectrometry for comprehensive PTM profiling

NFE2 undergoes several PTMs including phosphorylation on serine residues, which in undifferentiated erythrocytes leads to ubiquitination and protein degradation. It is also sumoylated, which is required for translocation to nuclear bodies, anchoring to gene loci, and transactivation of the beta-globin gene .

What are the best experimental controls for validating NFE2 antibody specificity?

To rigorously validate NFE2 antibody specificity:

• Positive controls: Use cell lines with known NFE2 expression (K-562, U-937 cells)

• Negative controls: Include NFE2 knockdown/knockout samples using RNAi or CRISPR-Cas9

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to block specific binding

• Cross-validation: Compare staining patterns with multiple NFE2 antibodies recognizing different epitopes

• Molecular weight verification: Confirm detection at the expected molecular weight (~45 kDa for NFE2)

A comprehensive validation study should demonstrate antibody specificity across multiple applications and conditions, as illustrated in antibody screening experiments comparing recombinant GST-NFE2 from E. coli with endogenous Nematostella NFE2 .

How can I optimize chromatin immunoprecipitation (ChIP) protocols for studying NFE2 binding sites?

For optimal NFE2 ChIP experiments:

• Crosslinking optimization: Test different formaldehyde concentrations (0.5-1%) and incubation times

• Sonication parameters: Adjust to achieve chromatin fragments of 200-500 bp

• Antibody selection: Use ChIP-validated NFE2 antibodies with demonstrated binding specificity

• Positive control regions: Include known NFE2 binding sites (e.g., UROS erythroid promoter at -27 bp, UROD promoter between -452 and -183 bp)

• Sequential ChIP: Consider for studying NFE2 complexes with other factors like MafG

This approach has successfully identified NFE2 binding to multiple heme biosynthetic enzyme promoters, including previously unknown targets UROS, UROD, and CPOX .

How should I interpret different subcellular localization patterns of NFE2 staining?

NFE2 subcellular localization is functionally significant and varies in different cell types and disease states:

• Normal pattern: Predominantly nuclear localization in mature erythroid cells

• Pathological patterns: Cytoplasmic mislocalization observed in primary myelofibrosis (PMF) patients

• Quantitative assessment: A threshold of 20% nuclear NFE2 staining has been cross-validated to discriminate between essential thrombocythemia (ET) and PMF

When analyzing subcellular localization:

• Use nuclear and cytoplasmic markers for co-localization studies

• Quantify the percentage of nuclear vs. cytoplasmic staining

• Compare with positive control tissues/cells

• Consider disease context when interpreting results

Mislocalization of NFE2 has demonstrated diagnostic value, with high interobserver concordance between pathologists (Spearman's rank correlation coefficient, 0.727) .

What strategies can resolve contradictory NFE2 antibody staining patterns in different experimental systems?

When facing contradictory NFE2 staining results:

• Epitope mapping: Different antibodies may recognize distinct epitopes affected by protein conformation or interaction partners

• Sample preparation variations: Compare fixation methods, antigen retrieval techniques, and blocking procedures

• Expression level differences: NFE2 expression varies by cell type; adjust antibody concentration accordingly

• Isoform specificity: Verify which NFE2 isoforms your antibody detects

• Post-translational modifications: Different antibodies may have varying sensitivities to phosphorylated, sumoylated, or ubiquitinated forms

For example, when screening NFE2 antibodies, researchers found that out of three antibodies tested, two had good specificity and signal at a 1:1000 dilution (Nrf_111, Nrf_243), while one showed poorer performance .

How can I address non-specific binding when using NFE2 antibodies in complex tissue samples?

To minimize non-specific binding:

• Optimization of blocking conditions: Test different blocking agents (BSA, normal serum, commercial blockers) and concentrations

• Antibody titration: Determine the minimum effective concentration to reduce background

• Pre-adsorption: Consider pre-adsorbing antibodies with tissues known to have non-specific binding sites

• Alternative detection systems: Compare direct vs. amplified detection methods

• Secondary antibody optimization: Test alternative secondary antibodies or detection formats

In IHC applications, heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with staining protocol can significantly improve specificity, as demonstrated with NFE2 antibody staining in human colon, testis, and tonsil tissues .

How can NFE2 antibodies be utilized in studying erythropoietic disorders and myeloproliferative neoplasms?

NFE2 antibodies offer valuable diagnostic and research applications in hematological disorders:

• Differential diagnosis: Quantitative NFE2 immunohistochemistry can reliably support differential diagnosis between essential thrombocythemia (ET) and primary myelofibrosis (PMF)

• Disease monitoring: Track NFE2 localization changes during disease progression

• Therapeutic response assessment: Monitor changes in NFE2 expression or localization following treatment

• Mechanistic studies: Investigate how NFE2 mislocalization contributes to disease pathophysiology

Studies have demonstrated that a threshold of 20% nuclear NFE2 staining discriminates between ET and PMF with high statistical significance, correctly classifying diagnostic bone marrow biopsies of MPN,U patients with 92% accuracy .

What experimental approaches can determine NFE2 protein interaction partners in different cellular contexts?

To identify NFE2 protein interactions:

• Co-immunoprecipitation with mass spectrometry: Immunoprecipitate NFE2 from cell/tissue homogenates followed by mass spectrometry analysis

• Proximity labeling approaches: BioID or APEX2 fusion proteins to identify proximal proteins

• FRET/BRET analyses: For studying dynamic interactions with known candidate partners

• ChIP-seq combined with proteomics: Identify co-factors at specific genomic loci

• Split-protein complementation assays: Visualize interactions in living cells

These approaches have identified NFE2 interactions with small MAF proteins (like MAFG) that are required for binding to the NF-E2 motif . More comprehensive mass spectrometry studies have identified numerous peptides associated with NFE2 through immunoprecipitation experiments .

How can evolutionary studies of NFE2 inform antibody selection for cross-species research?

For cross-species NFE2 research:

• Phylogenetic analysis: Understand evolutionary relationships between NFE2 proteins across species

• Domain conservation assessment: Target antibodies to highly conserved domains for cross-reactivity

• Epitope mapping: Align epitope sequences across species to predict cross-reactivity

• Validation in representative species: Empirically test antibodies in key model organisms

Phylogenetic and motif analyses have identified NFE2 proteins across diverse animal phyla, including chordates, arthropods, annelides, nematodes, echinoderms, molluscs, cnidarians, ctenophores, and sponges . These analyses reveal conserved domains that can guide antibody selection for evolutionary studies.

What knockdown/knockout validation strategies should be employed when studying NFE2 function with antibodies?

For rigorous validation of NFE2 antibodies using genetic approaches:

• siRNA knockdown: Transient reduction of NFE2 expression for short-term studies, as demonstrated in HEL cells using lentiviral shRNA constructs

• shRNA knockdown: More stable reduction for longer-term studies

• CRISPR-Cas9 knockout: Complete elimination of NFE2 expression

• Inducible systems: Temporal control of NFE2 expression for developmental studies

• Rescue experiments: Re-expression of NFE2 in knockout cells to confirm specificity

These approaches should include appropriate controls:

• Non-targeting siRNA/shRNA controls

• Quantification of knockdown efficiency by qPCR and Western blot

• Functional readouts of NFE2 activity, such as heme biosynthesis enzyme expression

How can NFE2 antibodies be used to study the relationship between NFE2 and heme biosynthesis?

To investigate NFE2's role in heme biosynthesis:

• ChIP-seq analysis: Map genome-wide NFE2 binding sites in erythroid cells, focusing on heme biosynthetic enzyme promoters

• Reporter gene assays: Test NFE2 transactivation of promoter constructs, as demonstrated with the UROS promoter

• Protein-DNA interaction analysis: EMSA or DNA pulldown assays to confirm direct binding

• Expression correlation studies: Combine NFE2 immunostaining with enzyme expression analysis

• Functional assays: Measure heme content after NFE2 modulation using microfluorometric assays

What methodological approaches are recommended for studying NFE2 in stress response and oxidative conditions?

To investigate NFE2 under stress conditions:

• Stress induction protocols: Expose cells to oxidative stress agents (H₂O₂, paraquat, hemin)

• Time-course analysis: Monitor NFE2 expression, localization, and PTMs over time

• Subcellular fractionation: Separate nuclear and cytoplasmic fractions to track translocation

• ChIP-seq under stress conditions: Map dynamic changes in NFE2 binding patterns

• Proteomic analysis: Identify stress-induced changes in NFE2 interaction partners

It's important to note that hemin induction increases NFE2 activity , and NFE2 may establish a positive feedback loop that secures a constant supply of both heme and globin for hemoglobin assembly during erythroid differentiation .