Phospho-ATM and p53 Activation Kit

The kit contains a monoclonal antibody that detects only the phosphorylated form of human ATM, anti-p53 polyclonal antibody, DyLight-conjugated Secondary Antibodies and various other reagents and buffers for immunofluorescence staining for high-content screening (HCS) assays.

Ataxia telangiectasia mutated kinase (ATM) is involved in cell-cycle check-point signaling and DNA repair. Mutation in the ATM gene leads to ataxia telangiectasia, an autosomal recessive human disease. ATM (350 kDa) is auto phosphorylated at Ser1981 upon induction of DNA double-strand breaks (DSBs) leading to rapid cellular check-point signaling and cell-cycle arrest. The tumor suppressor protein p53 is phosphorylated at Ser15 by ATM in response to DNA damage. ATM and p53 are important in protecting genomic integrity.

To measure phospho-ATM and p53 levels, A549 cells were treated with etoposide. Induction of DNA damage by etoposide leads to phosphorylation of ATM and an increase of p53 in the nucleus. The output parameter for this assay is the difference in nuclear and cytoplasmic intensity or average nuclear intensity for phospho-ATM and average nuclear intensity for p53. The assay was developed in A549 cells and optimized using the ArrayScan® HCS Reader and Compartmental Analysis Bioapplication Software Module. Cells stained using this kit also can be imaged using fluorescence or confocal microscopy.

Figure 2. Staining of p53 and phospho ATM. A549 cells were treated with vehicle (0.1% DMSO in media) or etoposide (50 µM) for 24 hours. Cells were stained according to the kit protocol and imaged using the ArrayScan HCS Reader.

Figure 3. Dose response curves for phospho-ATM and p53 treated with etoposide. Data represents mean ± SD from three plates (eight wells per 96-well plate per dose of etoposide).

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