Determination of Protein Lysine Acetylation by Western Blotting

About 5^∗10⁸ cells were harvested from mid exponential phase and extracted with BugBuster protein extraction reagent (Millipore) for 20 min at room temperature. 100 μL of SDS sample buffer (1% SDS, 2.5% 2-mercaptoethanol, 31.25 mM Tris-HCl pH 8.0, 5% glycerol, 0.0025% bromophenolblue) was added. 15 μl of extract was separated by SDS-PAGE using 12% polyacrylamide gels (Laemmli, 1970). Proteins were transferred to a Nylon membrane (0.2 μm, Millipore) by semi-dry blotting at 100 mA per gel for 20 min using transfer buffer (20% methanol, 1.3 mM SDS, 192 mM glycerol, 25 mM Tris). To check for equal protein amounts in the extracts, 5 μl of each extract was seperated by SDS-PAGE under the same conditions as above but stained with Coomassie brilliant blue.

Membranes were blocked with PBS 0.1% Tween 20 with 3% low fat milk powder in and probed with Acetylated Lysine Multi MAB (Cell Signaling Technologies) again in PBS 0.1% Tween with 3% low fat milk powder. As secondary antibody HRP labeled goat anti-rabbit secondary antibody (Sigma) was used in PBS 0.1% Tween 20. Signals were detected by using Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and a CCD camera (Intas).