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.
p21 mediates cell-cycle arrest in tandem with p53 (p53-dependant signaling). This p53-p21 axis is activated in response to multiple signals related to cell health and DNA damage. Some p21 promoters include pro-inflammatory pathway products, like JNK or p38, but also stress from radical oxygen species (ROS). The regulation of pro-apoptosis signaling, cell-cycle arrest, and DNA repair roles that p21 and p53 fulfill promote strong anti-proliferative processes and make them master effectors of several tumor suppressor pathways.
Physiologically relevant results fo fast flowing research - Flyers
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HTRF and WB compatible guidelines - Technical Notes
Protocol for tumor xenograft analysis with HTRF - Technical Notes
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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
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Analysis of a large panel of diverse biological samples and cellular models - Posters
One technology across all samples - Application Notes
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
Increased flexibility of phospho-assays - Application Notes
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
In collaboration with Bayer - Scientific Presentations
A fun video introducing you to phosphorylation assays with HTRF - Videos
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
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Learn how to reduce time and sample consumption - Application Notes
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