Phospho-ATM Activation Kit

The kit contains a primary monoclonal antibody that detects only the phosphorylated form of human ATM, a goat anti-mouse secondary antibody conjugated to DyLight™ 549 Fluorophore and various other reagents and buffers required for immunofluorescence staining for high-content screening (HCS) assays.

Ataxia telangiectasia mutated kinase (ATM, 350 kDa) 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 is auto phosphorylated at Ser1981 upon induction of DNA double-strand breaks (DSBs) leading to rapid check-point signaling. ATM kinase has several identified targets including H2AX, BRCA1, NBS1, Chk1, Chk2 and p53.

Phospho-ATM in the nucleus of A549 cells treated with etoposide was assayed using the Phospho-ATM Activation Kit, the Cellomics ArrayScan® HCS Reader and the Compartmental Analysis BioApplication Software Module. Induction of DNA damage by etoposide leads to phosphorylation of ATM. Cells labeled using this kit also can be imaged by fluorescence or confocal microscopy.

Figure 2. Staining of Phospho-ATM in A549 cells treated with vehicle (0.1% DMSO in media) (left) or with 50 µM etoposide for 3 hours (center). Cell were stained according to the kit protocol and imaged using a Cellomics ArrayScan® HCS Reader. Dose response curve for etoposide-treated A549 cells (bottom). Etoposide concentration is plotted against the difference between nuclear and cytoplasmic intensities for phospho-ATM. Data represents mean ± SD from three plates (eight wells per 96-well plate per dose of etoposide). EC₅₀ = 7.5 ± 1.1 µM.

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