Phospho-H2AX (Ser139) cellular kit
Fast and convenient, no wash phospho-H2AX (Ser139) assay kit
The HTRF Human p21 Detection kit is designed to detect the expression level of p21 in cell lysates.
p21, also known as cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1, is a key cyclin-CDK complex inhibitor that binds to cyclin-CK2, cyclin-CDK1, and cyclin-CDK4/6, and regulates their activity. p21 works in tandem with p53 to achieve downstream cell-cycle arrest following DNA damage, and is negatively regulated by ubiquitin-ligase-driven protein degradation.
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.
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.
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.
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One technology across all samples - Application Notes
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In collaboration with Bayer - Scientific Presentations
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