The wild-type and mutant plasmid constructs were used to generate 35S-methionine-labeled polypeptides using the TNT-coupled reticulocyte system (Promega). In vitro translated proteins were diluted in one bed volume (0.5 ml) of Column loading buffer [10 mM potassium phosphate (pH 6.2), 0.5% NP40, 10% glycerol, 1 mM DTT, aprotinin (1 mg/ml), pepstatin (1 mg/ml), leupeptin (1 mg/ml)], and applied to native DNA cellulose columns (Pharmacia). The protein sample was passed through the column twice. The columns used were Poly-Prep Chromatography Columns (Bio Rad catalog number 731-1550). Unbound material is designated flow-through (FT). The columns were then washed multiple times with 1.0 bed volume column-loading buffer containing 50 mM NaCl (these are the 50 mM wash fractions), and eluted stepwise with column-loading buffer adjusted to contain increasing concentrations of NaCl from 100 to 800 mM, as indicated in the text. Fractions were analyzed by SDS–PAGE. The signal on the dried gel was quantified using a phosphorimager (Fuji) and associated software.
