HTRF human and mouse total NRF2 assay principle

The HTRF Human/mouse Total NRF2 assay quantifies the expression level of NRF2 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Human Total NRF2 assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of NRF2 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

Human and mouse Total NRF2 two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Human/mouse Total NRF2 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Human and mouse Total NRF2 one-plate assay protocol

Detection of Human NRF2 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

NRF2 release with compounds

Hep-G2 cells were plated at 200,000 per well in a 96-well culture-treated plate in complete culture medium, and incubated overnight at 37°C, 5% CO2. They were treated with increasing concentrations of tBHQ, Ki696, and Thapsigargin for 16h at 37 °C, 5% CO2, and then lysed with 50 µl of supplemented lysis buffer #1 (1X) for 30 min at RT under gentle shaking.

After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total NRF2 detection reagents were added. The HTRF signal was recorded after 18h of incubation at room temperature.

As expected, these compounds induced the release of NRF2 from Keap1 with a dose-dependent increase in the NRF2 signal level with different EC50: 38 nM for Ki696, 0.3 µM for thapsigargin, and 29 µM for tBHQ.

Assessment of NRF2 levels in various human and mouse cell lines

Adherent human & mouse cells Hep-G2, HEK-293, HAP1, and NIH 3T3 were seeded at 100,000 or 200,000 cells / well in a 96-well microplate. After a 24h incubation, the cells were treated with tBHQ for 16h at 37°C, and then lysed with supplemented lysis buffer #1 at RT under gentle shaking. Next, 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF total NRF2 detection reagents. The HTRF signal was recorded after an overnight incubation.

The HTRF Total NRF2 assay efficiently detected NRF2 protein in various cellular models expressing different levels of the protein.

Assay specificity of HTRF Total NRF2 assay

HAP1 and HAP1 NRF2 KO cells (from Biolegend) were cultured in a T175 flask in a complete culture medium for 24h at 37°C, 5% CO2. The cells were lysed with 3 mL of supplemented lysis buffer # 1 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were then transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF total NRF2 detection reagents. The HTRF signal was recorded after an overnight incubation at RT..

No signal was detected for the HAP1 cells KO NRF2, whereas a signal was detected in the HAP1 wt, thus proving the specificity of the assay for NRF2 protein.

HTRF Human Total NRF2 assay compared to Western Blot

Hep-G2 cells were cultured in a T175 flask in a complete culture medium for 24h at 37°C, 5% CO2. The cells were treated with 100 µM of tBHQ compound for 16h at 37°C, 5% CO2, and then lysed with 3 mL of supplemented lysis buffer #1 (1x) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer #1 (1x), and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF (h/m) Total NRF2 detection reagents.

Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

In these conditions, the HTRF Total NRF2 assay was 4 times more sensitive than the Western Blot technique.

NRF2 signaling pathway

The overproduction of reactive oxygen species (ROS) generates oxidative stress in cells, which results in various pathophysiological conditions, especially cancers and neurodegenerative diseases (NDD). The Keap1-Nrf2 [Kelch-like ECH-associated protein 1-nuclear factor (erythroid-derived 2)-like 2] regulatory pathway plays a central role in protecting cells against oxidative and xenobiotic stresses.

NRF2, which is controlled by KEAP1, is phosphorylated under cell stress, and this allows its release from KEAP1 protein. Free NRF2 translocates to the nucleus. The Nrf2 transcription factor activates the transcription of several cytoprotective genes, including p62SQSTM1, that have been implicated in protection from cancer and NDD. These 2 proteins are important regulators of the autophagy pathway.