Cells were lysed by sonication on ice water (2 × 20 sec, Branson sonifier 250, 3 mm disruptor horn, output level 2, constant), and the lysate cleared by centrifugation

at 14000 rpm, 18°C for 20 min in a tabletop centrifuge. A cellulose column was prepared by pipetting 30 mg Avicell PH-101 (Fluka) resuspended in 300 μlCFE into a Mobicol empty spin column (MoBiTec). The column was centrifuged (300 × g, 1 min, RT), washed with 600 μlCFE to remove fines and centrifuged again. The cleared lysate was RG7112 applied to the column in 600 μlportions and the cellulose resuspended by pipetting up and down. After 1 min incubation at room temperature, the column was centrifuged (300 × g, 1 min, RT) and the flow-through discarded. The cellulose was washed three times with 600 μlCFE + 0.5% NP40 (Roche) and once with CFE. After each washing step the column was

centrifuged (300 × g, 1 min, RT) and the flow-through discarded. An additional centrifugation (770 × g, 1 min, RT) was performed after the last washing step to reduce the amount of retained buffer. For elution, 600 μl ethylene glycol (Merck, Darmstadt) were applied to the column, the cellulose resuspended, and the column centrifuged. Eluted proteins were precipitated with TCA. For this, an equal volume of 20% (w/v) TCA was added to the eluate, the mixture incubated on ice for 30 min and centrifuged at 14000 rpm, 4°C, 30 min. Finally, the pellet was washed 2-3 times with ice-cold 50% (w/v) acetone. For SILAC-based one-step bait-fishing experiments the above protocol was modified as follows: The bait expression strain and the bait-control strain were precultured in 35 ml complex medium containing 0.15 μgm l −1 novobiocin at 37°C on a GSK923295 clinical trial shaker (150 rpm) until an O D 600of 0.5-1.0 was reached. Five hundred microliters of these

cultures were used to inoculate second precultures that were grown under identical conditions to an O D 600of 0.8-1.0. The second precultures were used to inoculate 100 ml synthetic medium Edoxaban containing 13C6-leucine for the bait expression strain and 12C6-leucine for the bait-control strain at an O D 600 of 0.01; the inoculum was adjusted to 1.5 ml with complex medium before addition to the 100 ml medium. The main cultures were incubated on a shaker (110 rpm) at 37°C in the dark until they reached an O D 600 of 0.8. Cells were harvested by centrifugation (8000 rpm, 15°C, 15 min) and pellets resuspended in 1 ml CFE + PI. Cell lysate and cellulose columns were prepared as described above. Three hundred microliters lysate from each culture were applied to the column, the cellulose resuspended, and after 1 min incubation the column centrifuged (300 × g, 1 min, RT). This step was repeated twice, followed by washing, elution, and protein this website precipitation as described. Two-Step bait-fishing experiments were performed with the following modifications: Hbt.salinarum R1 was precultured twice in 35 ml complex medium at 37°C on a shaker (110 rpm) until an O D 600 of 0.5-1.0 was reached.