This activity of COST Action CA20121 (BenBedPhar) consolidates current best practices, caveats, and method-focused guidance for studying NRF2 across non-communicable diseases. It emphasizes standardization in experimental design, data acquisition, and interpretation, particularly in assays used to quantify NRF2 levels/activity (e.g., antibody-based detection, target-gene readouts) and in workflows that assess the KEAP1/NRF2 axis in cellular, tissue, and clinical samples.
Recent methodological guidance highlights critical issues for antibody-based detection of NRF2, including expected migration patterns in SDS-PAGE/Western blots, antibody specificity pitfalls (e.g., cross-reactivity with proteins such as calmegin), and validation steps (knockdown/overexpression, multiple antibodies, and orthogonal readouts). These recommendations urge caution in band assignment, advocate the use of validated monoclonals, and encourage reporting of gel conditions and controls to ensure reproducibility. ScienceDirect+2PubMed+2
Complementary, field-level practice notes synthesize “what often goes wrong” in NRF2/KEAP1 research, covering experimental variables (stressors, electrophiles), context-dependence of pathway readouts, and interpretation of indirect reporters, so that laboratories can avoid common artifacts, adopt transparent reporting, and harmonize protocols across sites. PubMed
Together, these sources support a practical framework for BenBedPhar partners: (i) adopt validated detection reagents and orthogonal confirmation of NRF2 activity; (ii) standardize sample handling, gel systems, and loading controls; (iii) pair protein detection with target-gene/protein panels for functional activity; and (iv) document assay conditions and quality controls to enable cross-study comparability and meta-analysis across the network. This activity strengthens the Action’s methodological harmonization and translational readiness for NRF2-focused investigations in NCDs.
