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Bacterial pellets were sonicated at 70% amplitude, 2 s on5 s off for 5 min on ice in lysis buffer (20 mM Tris-HCl, pH 7.2, 500 mM NaCl, 10 mM imidazole, 5% glycerol, 0.5 mg ml 1 lysozyme (Thermo, 89833), 0.5% octyl-B-D-thioglucopyranoside (CHEM-IMPEX, 21810), 2 mM PMSF (Roche) and 2 mM -mercaptoethanol (Sigma, M3148), and the pH was adjusted to 8.0 with NaOH), after which, Benzonase (~25 U ml 1 ), MgCl 2 (1 mM) and CaCl 2 (1 mM) were added and the lysate was incubated on ice for 15 min

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[72] 20042009: Queen + Paul Rodgers [edit] At the end of 2004, May and Taylor announced that they would reunite and return to touring in 2005, with Paul Rodgers, the founder and former lead singer of Free and Bad Company