ATM is a high MW protein of ~350kDa and as such, conditions may require optimization. 100-150µg of cell lysate is loaded on 5% acrylamide gels (until 200kDa marker is halfway through the gel) and transferred to nitrocellulose using semi-dry transfer at 9V constant voltage for 2 hours. Detection should be done using HRP-conjugated anti-rabbit IgG. This antibody may detect several smaller non-specific bands present in both ATM mutant and wild type cells. Therefore, careful titering of antibody concentrations is recommended.