HTRF Human p21 assay principle

The HTRF Human p21 assay quantifies the expression level of human p21 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. In presence of p21 in a cell lysate, 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.

HTRF Human p21 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 p21 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

p21 activation in Tenovin-1-stimulated MCF-7

MCF-7 cells were seeded at 50,000 cell per well under 100 µl in a 96-well plates in complete culture medium and incubated overnight at 37°C, 5% CO2. After incubation, cells were treated with a dose-response of the p53 activator Tenovin-1, and incubated for 24h at 37°C, 5% CO2.

After incubation, culture medium was removed and cells were lysed with 50 µL of supplemented lysis buffer #2 at 1X for 30 minutes at RT under gentle shaking.

16 µL of lysate were transferred into a low volume white microplate before the addition of 2 µL of the HTRF d2 detection reagent and 2 µL HTRF Eu-K detection reagent. The HTRF signal was recorded after a 4h incubation.