Following centrifugation at 10,000×g at 4 °C for 10 min, supernatants were saved (periplasmic fraction, P1), and pellet were resuspended in 0.6 mL of ice-cold 5 mM MgSO4 (hypotonic solution) and shaked slowly for 10 min on ice.
After centrifugation at 18,000 g for 30 min, supernatants were saved and the pellet was solubilized in 0.1 M sodium carbonate (pH 11.5) and incubated on ice for an hour.
Fifty microliters of sonicated cell extracts were centrifuged at 4°C at 15,000 × g for 30 min, the supernatants were saved and the pellets were resuspended in 50μL of denaturation buffer (50 μmmol/l Tris-Cl pH8, 0.1 mol/l β-mercaptoethanol, 3% SDS).
Cell nuclei were collected by centrifugation at 5,000 rpm for 15 min, and the supernatants were saved as cytoplasmic protein preparation.
After centrifugation (1,000 g, 10 min, 4°C), supernatants (SN1) were saved.
After sonication, samples were pelleted via centrifugation (10,000 x g, 30 min; 4°C) and supernatants were saved.
The sonicates were centrifuged at 10 000 r.c.f. for 10 min and the supernatants were saved for biochemical analysis.
The homogenates were centrifuged two times at 20 000 × g at 4 °C for 15 min, and the supernatants were saved as total proteins.
After sonication, lysates were centrifuged at 16,000 g for 20 min, and these supernatants were saved as Triton X-100-insoluble fractions.
