Mouse anti ack antibody

Cells from 1 ml culture were centrifuged and 1X LDS sample buffer with reducing agent (Thermo Fisher) was added to the cell pellet, sufficient to give an OD₆₀₀ of 2.5. Samples were boiled and resolved on a NuPAGE 10% Bis-Tris Gel in MOPS SDS Running Buffer from Thermo Fisher. Proteins were transferred onto a nitrocellulose membrane with an iBlot gel transfer system. Lysine-acetylated proteins were detected with mouse anti-AcK antibody (Cell Signaling Technology, cat. # 9681S) diluted 1/1000 in TBS-T with 5% of BSA, incubated overnight at 4°C and developed with peroxidase conjugated anti-mouse antibody. The signal was detected with a chemiluminescent reaction, using ECL^TM Western Blotting kit from GE Healthcare, and detected by a BioRad Molecular Imager ChemiDoc XRS System.

Cells were grown by duplicate in minimal media with aeration at 37°C up to an OD₆₀₀ of 0.1. Then,1 ml culture was centrifuged, and 1X Sample Buffer (Thermo Fisher) was added to the cell pellet. Samples were boiled and resolved on a NuPAGE 10% Bis-Tris Gel in MES SDS Running Buffer from Thermo Fisher. Proteins were transferred onto a nitrocellulose membrane with an iBlot gel transfer system. Crp and RpoA were detected with α-Crp, α-RpoA mouse antibody (Biolegend) diluted 1/5000 in PBS-T with 2.5% of Milk. To detect lysine-acetylated proteins mouse anti-AcK antibody (Cell Signaling Technology) diluted 1/1000 in TBS-T with 5% of BSA was used. IRDye 800CW Donkey anti-mouse (Li-Cor) was used as secondary antibody. The signal was detected with Odyssey CLX System from Li-Cor, and the intensity of the different bands was quantified using the software Image Studio Lite from Li-Cor.