Analytical gel filtration chromatography

Purified CsrA and Re-CsrA were individually mixed with CesT with a molar ratio of 1.2:1, followed by incubation at 4 °C for 2 h. The samples, including the individual CsrA, Re-CsrA, and CesT proteins, as well as the CesT/CsrA and CesT/Re-CsrA mixtures, were loaded onto a calibrated Superdex 200 Increase 10/300 GL (GE Healthcare) column, respectively. The UV-280 curves were recorded and overlaid. The pooled samples were further analyzed by SDS-PAGE (15% gel) and stained with Coomassie blue.

For CesT and CsrA mutants, the purified proteins and their respective wild-type proteins were individually loaded on the Superdex 200 Increase 10/300 GL (GE Healthcare) column for comparative analytical gel filtration analyses. The UV-absorbance curves were recorded at 280-nm for all the proteins except for CsrA-C_del, whose absorbance chromatograph was recorded at 215-nm due to the lack of any aromatic residues after deletion of the C-terminal helix. The curves were then overlaid for comparison.