Affinity Purification and Radiolabeling of Proteins

Frozen or fresh cultures of staphylococci were washed and blocked with ice-cold 4% PBSB. The target protein or lysate was added in ice-cold 4% PBSB or lysis buffer, and incubated at 4 °C for 60 min. After washing thrice by three minutes centrifugation at 6000 rpm and 4 °C, bound proteins were eluted with 0.2 M glycine (pH 2.2) and neutralized by addition of 1 M Tris (pH 9.1) or with 1% SDS. Radiolabeling was performed and the lysates prepared as recently reported [72 (link)]. Briefly, cells were labeled at 37 °C using methionine- and cysteine-free medium supplemented with 10 mCi/mL [³⁵S]Met/Cys (PerkinElmer, Waltham, MA, USA).