Cycloheximide chase analysis of protein stability

Cycloheximide (CHX) was added to mid-log phase cells at a final concentration of 35 μg/ml. Cell aliquots were taken hourly, washed and freeze in liquid nitrogen. To prepare cell lysates, cells were re-suspended in 200 μl 2% sodium dodecylsulphate (SDS) and were lysed by adding glass beads (Sigma) followed by four working cycles of 1 min in a bullet blender (Next Advance). The lysates were cleared by two centrifugations of 5 and 15 min at 15 000 g at 4°C. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and were analyzed by western blot with the indicated antibodies.