Ku70/80 Activation Kit

The kit contains a primary monoclonal antibody specific for human Ku70/80 heterodimers, a goat anti-mouse secondary antibody conjugated to DyLight™ 549 Fluorophore, and the various other reagents and buffers required for immunofluorescence labeling of Ku70/80 for high-content screening (HCS) assays.

The Ku70/80 heterodimer plays an important role in DNA double-strand break (DSB) repair. During DSB repair by non-homologous end joining (NHEJ), Ku70/80 binds with high affinity to DNA ends of a DSB and then recruits the catalytic domain of DNA protein kinase (DNAPK). The formation of DNAPK complex at the site of DSBs results in the recruitment of other repair proteins to ligate the broken ends. Recently, it has been shown that Ku70/80 has a role in ATM-dependent activation of ATR during DNA DSB damage response.

Increased expression of Ku70/80 heterodimers in the nucleus after treating A549 cells with 75 µM etoposide to induce DNA damage was quantitatively assayed using the Cellomics Ku70/80 Activation Kit, the Cellomics ArrayScan HCS Reader and Compartmental Analysis Bioapplication Software Module. Induction of DNA damage by etoposide leads to increase in Ku70/80 nuclear staining.

Figure 2. Increased nuclear Ku70/80 heterodimer formation in A549 cells upon treatment with etoposide. A549 cells were treated with vehicle (0.1% DMSO in media) or 75 µM etoposide in media for 24 hours, then fixed and stained for Ku70/80 and nuclei (Hoechst 33342 Dye) according to the kit protocol. The cells were imaged using a Cellomics ArrayScan HCS Reader. Dose response curve for etoposide-treated A549 cells. Etoposide concentration is plotted against the difference between nuclear and cytoplasmic intensities for Ku70/80. Data represents mean ± SD from three plates (eight wells per 96-well plate per dose of etoposide). EC₅₀ = 18.15 ± 2.2 µM.

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