Cells were harvest by centrifugation for 10 min at 4 ºС at 12000Xg.
The supernatant was discarded, and the pellet was washed twice with pH8.0 10mM Tris-HCl buffer, following centrifugation, cells were suspended in lysis buffer (containing 10mM tris-HCl, 1%SDS, 0.1M DTT), sonicated for 30s with pulse 5 times, the supernatant was collected by centrifugation.
To each tube, chilled 100% trichloroacetic acid (TCA) was added to achieve ∼25% TCA as final concentration.
Tubes were inverted gently and kept at -10 ºС overnight to precipitate proteins present in the solution.
Samples were centrifuged at 20800× g for 20 min to obtain a concentrated protein pellet and the supernatant (containing detergent and other contaminants) was discarded.