Dynabeads kilobasebinder magnetic streptavidin beads

Bio-Flag-ATM was incubated with varying amounts of MRN, GST-p53 (1–102) peptide, or homeodomain protein, as indicated in figure legends. Proteins were incubated at room temperature for 30 min. Then, 2.5 mL of Dynabeads kilobaseBINDER magnetic streptavidin beads (Invitrogen) were pre-washed with Buffer A containing 100 mM NaCl and 1 mg/mL BSA and incubated with the protein mixture for 15 min at 4°C in the presence of 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (Sigma). Biotinylated protein and associated factors were isolated using a magnet and were washed three times with Buffer A containing 100 mM NaCl and 0.1% CHAPS. Beads were resuspended in 2.53 SDS loading buffer and boiled for 10 min before loading onto a 9% denaturing SDS-PAGE gel. Proteins were transferred for 3 hr at 400 mA and analyzed by western blotting as indicated in figure legends.