Transam nrf2 transcription factor assay kit

Nrf2 activation assay was carried out as described in the company’s protocol (TransAM Nrf2 Transcription Factor Assay Kit, Cat No. 50296, Active motif, Carlsland, CA, USA) and as described in our published protocol [11 (link),67 (link)]. Briefly, 10 µg of nuclear extract (up to 10 µL diluted with complete lysis buffer) prepared from H₂O₂ exposed LECs was added to the strips well following the addition of 40 µL binding buffer containing 20 pmol of the wild-type or mutated consensus oligonucleotide to each well. The plate was incubated for 1 h at room temperature (RT). Then, 100 µL primary antibody (1:1000 dilution) was added after 3 washes and then incubated at RT for 1 h. 100 µL of diluted anti-rabbit HRP conjugated antibody (1:1000 dilution) was added and incubated for 1 h at RT. Then, 100 µL of developing solution was added to wells after washing and incubated for 2 to 10 min in the dark. Finally, 100 µL of stop solution was added, and Optical Density (O.D.) was measured at 450 nm.

Nrf2 activation assay was performed according to manufacturer′s protocol (TransAM Nrf2 Transcription Factor Assay Kit, Cat No 50296, Active motif, Carlsland, California, USA). In brief, 10 µg of nuclear extract (up to 10 µL diluted with complete lysis buffer) prepared from SFN treated hLECs added to the strips well, following the addition of 40 µL complete binding buffer contains 20 pmol of the wild-type and/or mutated consensus oligonucleotide to each sample well. For blank well, 10 µL of complete lysis buffer were used. The plate was incubated for 1 h at room temperature (RT) with mild agitation. 100 µL primary antibody (1:1000 in 1× antibody binding buffer) added after three washes with 1× washing buffer and incubated at RT for 1 h without agitation. 100 µL of diluted anti-rabbit HRP (horseradish peroxidase)- conjugated antibody (1:1000 dilution in 1× antibody binding buffer) after three washing was added and incubated for 1 h at RT. 100 µL of developing solution was added to wells after four washing and incubated at RT in dark for 2 to 10 min. Finally, by addition of 100 µL of stop solution, optical density (O.D.) was recorded at absorbance 450 nm.