Phospho-p62/SQSTM1 (Ser403) Cellular Kit
Simple and robust detection kit for Phospho-p62/SQSTM1 (Ser403)
This cell-based HTRF kit enables the quantitative detection of Total NRF2 in human and mouse cell lysates.
The HTRF Human/mouse NRF2 detection assay monitors NRF2 expression in endogenous NRF2 in various cells.
The overproduction of reactive oxygen species (ROS) generates oxidative stress in cells. NRF2 is under the control of KEAP1. Under cell stress, NRF2 is phosphorylated allowing its release from KEAP1 protein. NRF2 then 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.
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.
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.
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.
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.
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.
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.
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.
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.
Mastering the art of cell signaling assays optimization - Guides
physiologically relevant results fo fast flowing research - Flyers
Insider Tips for successful sample treatment - Notes techniques
HTRF and WB compatible guidelines - Notes techniques
Protocol for tumor xenograft analysis with HTRF - Notes techniques
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
A solution for phospho-protein analysis in metabolic disorders - Posters
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
PI3K/AKT/mTor translational control pathway - Posters
Analysis of a large panel of diverse biological samples and cellular models - Posters
One technology across all samples - Notes d'application
Tumor xenograft analysis: HTRF versus Western blot - Notes d'application
Valuable guidelines for efficiently analyzing and interpreting results - Notes d'application
Increased flexibility of phospho-assays - Notes d'application
Analyse of PI3K/AKT/mTor translational control pathway - Notes d'application
In collaboration with Bayer - Présentations scientifiaues
A fun video introducing you to phosphorylation assays with HTRF - Vidéos
Seeding and lysing recommendations for a number of cell culture vessels. - Notes techniques
Learn how to reduce time and sample consumption - Notes d'application
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Notes d'application
64NRF2TPEG Lot 01A - Rapports de contrôle qualité
64NRF2TPEH Lot 01A - Rapports de contrôle qualité
Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.Let's find your reader