NIFU2 Antibody

What is NRF2 and how does it typically appear in SDS-PAGE?

NRF2 (Nuclear factor erythroid 2-related factor 2) is a transcription factor involved in cellular stress response. In SDS-PAGE, human NRF2 typically migrates above 100 kDa, with the exact position depending on the percentage of the gel used. In 8% Tris-glycine gels, monoclonal anti-NRF2 antibodies typically detect NRF2 as three bands migrating between 100 and 130 kDa . This migration pattern is significantly higher than the theoretical molecular weight calculated from the amino acid sequence, which is a common property of many transcription factors due to post-translational modifications and intrinsically disordered regions.

Why is antibody validation critical for NRF2 research?

Antibody validation is particularly crucial for NRF2 research because recent mass spectrometry studies have identified that several commercial anti-NRF2 antibodies bind to calmegin, an ER-residing chaperone that co-migrates with NRF2 in SDS-PAGE . This cross-reactivity can lead to misinterpretation of experimental results, particularly when analyzing NRF2 levels in different cellular compartments. Proper validation through knockdown experiments, stimulation with NRF2 activators (such as tert-BHQ), and translation inhibition tests can help distinguish true NRF2 signals from calmegin.

What are the common applications for NRF2 antibodies in research?

NRF2 antibodies are primarily used in western blotting, immunoprecipitation, and immunofluorescence studies to investigate:

• Nuclear translocation of NRF2 upon stress conditions

• Protein-protein interactions involving NRF2

• Changes in NRF2 stability and degradation

• Subcellular localization of NRF2

• Effects of drug treatments on NRF2 pathway activation

For western blot analysis, antibodies such as anti-NRF2 [EP1808Y] (Abcam), anti-NRF2 (D1Z9C) XP (Cell Signaling Technology), and anti-NRF2 (E5F1A) (Cell Signaling Technology) have been used, though with varying degrees of specificity .

How can researchers distinguish between NRF2 and calmegin in western blot analysis?

Distinguishing between NRF2 and calmegin in western blots requires strategic experimental approaches:

• Translation inhibition: Treatment with translation inhibitors like emetine (20 μM for 2 hours) will decrease NRF2 levels but not affect calmegin levels .

• NRF2 activation: Treatment with NRF2 activators such as tert-BHQ (50 μM for 4-5 hours) increases NRF2 levels while calmegin remains unchanged .

• Subcellular fractionation: NRF2 is predominantly nuclear upon activation, while calmegin is primarily found in the cytoplasmic/ER fraction . Proper fractionation can help differentiate between these proteins.

• Knockdown experiments: siRNA-mediated knockdown of NRF2 will reduce NRF2 bands but not affect calmegin bands .

• Mass spectrometry verification: For definitive identification, immunoprecipitated proteins can be analyzed by mass spectrometry as demonstrated in recent studies .

Which NRF2 antibodies show highest specificity in immunofluorescence studies?

Based on validation studies with NRF2 knockdown and tert-BHQ treatment, the Cell Signaling E5F1A antibody demonstrates high specificity for nuclear NRF2 in immunofluorescence applications . This antibody shows weak nuclear signals under basal conditions but strong nuclear accumulation upon tert-BHQ treatment, which is abrogated by NRF2 knockdown .

Interestingly, some antibodies that cross-react with calmegin in western blots can still be useful for detecting nuclear NRF2 in immunofluorescence. For instance, the Abcam EP1808Y antibody shows specific nuclear NRF2 accumulation upon tert-BHQ treatment that is eliminated by NRF2 knockdown, suggesting it can be used for detecting nuclear NRF2 despite its cross-reactivity with calmegin in western blots .

What sample preparation methods optimize NRF2 detection?

Optimal sample preparation for NRF2 detection includes:

• Protein extraction: Use of IP buffer (25 mM Tris pH 7.5, 150 mM NaCl, 0.5% TritonX-100) followed by sonication and centrifugation has proven effective for NRF2 immunoprecipitation studies .

• Gel selection: 8% Tris-glycine SDS-PAGE gels provide optimal separation for NRF2, which migrates between 100-130 kDa .

• For immunofluorescence: Fixation with ice-cold 4% para-formaldehyde for 10 minutes, followed by permeabilization with 0.2% Triton X-100 for 5 minutes has been successful. Blocking with 5% FBS or 10% goat serum improves specificity .

• Signal amplification: For weak signals, tyramide-based signal enhancement may be necessary, especially with the Cell Signaling E5F1A antibody .

What controls are essential when working with NRF2 antibodies?

Essential controls for NRF2 antibody experiments include:

• Positive controls:

  • Treatment with known NRF2 activators (tert-BHQ, 50 μM for 4-5 hours)

  • Overexpression of NRF2 in appropriate cell lines

• Negative controls:

  • siRNA or shRNA-mediated knockdown of NRF2

  • Translation inhibition (emetine, 20 μM for 2 hours) to reduce NRF2 levels

  • Ideally, NRF2 knockout cell lines when available

• Specificity controls:

  • Analysis of calmegin expression

  • Subcellular fractionation to confirm proper localization

  • Secondary antibody-only controls for immunofluorescence

• Technical controls:

  • Loading controls (β-actin, tubulin)

  • Nuclear fraction controls (lamin A)

How should researchers validate antibody specificity for NRF2?

A comprehensive validation strategy for NRF2 antibodies should include:

• Knockdown/knockout verification: Confirm signal reduction with NRF2 siRNA or in NRF2 knockout cells.

• Pharmacological manipulation:

  • Increased signal with NRF2 activators (tert-BHQ)

  • Decreased signal with translation inhibitors (emetine)

• Mass spectrometry confirmation: Immunoprecipitate protein bands of interest and identify by LC-MS/MS to confirm NRF2 presence and detect potential cross-reactive proteins .

• Comparative antibody analysis: Test multiple antibodies with different epitopes to confirm consistent detection patterns.

• Cell fractionation: Verify expected subcellular distribution of the detected protein.

What are the optimal conditions for immunoprecipitation of NRF2?

For successful NRF2 immunoprecipitation:

• Cell preparation: Use 10 × 10^6 cells per condition for adequate protein yield .

• Lysis conditions: IP buffer (25 mM Tris pH 7.5, 150 mM NaCl, 0.5% TritonX-100) with sonication and centrifugation .

• Antibody selection: The Abcam EP1808Y antibody (2 μg per sample) has been successfully used for NRF2 immunoprecipitation .

• Incubation conditions: Overnight incubation at 4°C provides optimal binding.

• Bead selection: 50 μl of protein G magnetic beads are suitable for pulldown .

• Washing and elution: Multiple washes with IP buffer and PBS, followed by elution in 2× Laemmli buffer at 50°C for 10 minutes .

• Gel separation: Use 8% SDS-PAGE for optimal separation of the 100-130 kDa range containing NRF2 .

Why might multiple bands appear in NRF2 western blots?

Multiple bands in NRF2 western blots can result from:

• Cross-reactivity: Many anti-NRF2 antibodies cross-react with calmegin, which co-migrates with NRF2 in SDS-PAGE. This is particularly problematic as calmegin often gives stronger western blot signals than NRF2 .

• Post-translational modifications: NRF2 undergoes various post-translational modifications including phosphorylation and ubiquitination that can affect migration.

• Degradation products: NRF2 has a short half-life and may appear as degradation fragments.

• Alternative splicing: Different NRF2 isoforms may be detected.

To distinguish between these possibilities:

• Compare signal patterns after NRF2 knockdown

• Use tert-BHQ treatment to stabilize NRF2

• Perform translation inhibition with emetine to differentiate NRF2 (decreases) from calmegin (stable)

What strategies can improve specificity in NRF2 immunofluorescence?

To improve specificity in NRF2 immunofluorescence:

• Antibody selection: The Cell Signaling E5F1A antibody shows high specificity for nuclear NRF2 .

• Signal amplification: For weak signals, use tyramide-based signal amplification (Tyramide SuperBoost kit) with 7-minute incubation with tyramide reagent .

• Validation controls: Include NRF2 knockdown samples and tert-BHQ-treated samples as negative and positive controls, respectively .

• Blocking optimization: Use 5% FBS or 10% goat serum for optimal blocking .

• Confocal microscopy: Use confocal microscopy with z-stack analysis to accurately determine subcellular localization.

• Nuclear counterstaining: Use DAPI (300 nM) to clearly define nuclear boundaries .

How can researchers interpret conflicting western blot and immunofluorescence results?

When western blot and immunofluorescence results seem contradictory:

• Consider cross-reactivity: Antibodies may detect different proteins in different applications. For example, some antibodies that cross-react with calmegin in western blots may still specifically detect nuclear NRF2 in immunofluorescence .

• Evaluate subcellular localization: In immunofluorescence, focus on nuclear localization for NRF2, as calmegin is primarily cytoplasmic/ER-associated .

• Verify with fractionation: Perform cell fractionation followed by western blot to confirm the localization patterns observed in immunofluorescence.

• Use multiple antibodies: Compare results with multiple antibodies having different epitopes.

• Functional validation: Use NRF2 activators (tert-BHQ) and knockdown approaches to confirm the functional identity of the detected protein .

How can mass spectrometry complement antibody-based NRF2 detection?

Mass spectrometry provides powerful verification for antibody-based detection of NRF2:

• Protein identification: LC-MS/MS can definitively identify proteins immunoprecipitated by anti-NRF2 antibodies, distinguishing between NRF2 and cross-reactive proteins like calmegin .

• Protocol overview:

  • Immunoprecipitate proteins using anti-NRF2 antibodies

  • Separate by SDS-PAGE and stain (e.g., with Flamingo stain)

  • Excise gel regions containing proteins of interest

  • Perform in-gel tryptic digestion

  • Analyze peptides by LC-MS/MS

• Quantitative analysis: Extract chromatograms of representative peptides using tools like Skyline to confirm reliable detection and quantification .

• Coverage assessment: Analyze peptide coverage across protein sequences to distinguish between similar proteins .

What specific mass spectrometry protocols have been validated for NRF2 detection?

The following validated mass spectrometry protocol has been used successfully for NRF2 detection:

• Sample preparation: Immunoprecipitate NRF2 from control cells, emetine-treated cells (20 μM, 2h), and tert-BHQ-treated cells (50 μM, 4h) .

• Gel separation: Separate immunoprecipitates on 8% Tris-glycine SDS-PAGE and stain with Flamingo .

• Sample processing:

  • Excise gel pieces containing 100-130 kDa proteins

  • Fragment to 3 mm pieces

  • Rinse with buffers (25% Acetonitrile/75% 50 mM NH₄HCO₃ and 50% Acetonitrile/75% 50 mM NH₄HCO₃)

  • Reduce with 20 mM DTT in 50 mM NH₄HCO₃ (30 min)

  • Alkylate with 60 mM iodoacetamide (60 min, dark)

• Digestion and extraction:

  • Digest with 10 ng/μl trypsin in 50 mM NH₄HCO₃ overnight

  • Extract peptides with 5% TFA and acetonitrile

  • Dry extracted peptides under vacuum

• LC-MS/MS analysis: Analyze using appropriate LC-MS/MS protocols to identify NRF2 and potential cross-reactive proteins .