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. Author manuscript; available in PMC: 2023 Sep 27.
Published in final edited form as: Microbiology (Reading). 2018 Nov 20;165(1):15–25. doi: 10.1099/mic.0.000732

Fig. 5. SrcA interacts directly with SteD (a), Indicated Salmonella strains were grown in minimal medium pH 5.0 for 5h.

Fig. 5

Cells were then pelleted and frozen overnight. Cells were resuspended in lysate buffer (500 mM NaCl, 1 mM PMSF in PBS) and lysed by sonication, followed by addition of Triton X-100. Proteins were immunoprecipitated with anti-FLAG M2 affinity gel and samples were analysed by immunoblotting with antibodies against HA, FLAG and DnaK. Light chain indicates the light chain of the anti-FLAG antibody in the affinity gel, which is also detected by the secondary antibody in the Western blot. (b) A complex of SteD and SrcA was expressed in E. coli from a single plasmid under the lac promotor and purified to homogeneity on NiNTA beads. The size of the complex was examined using size exclusion chromatography. The obtained peak was analysed using SDS-PAGE and subsequent mass spectrometry of the protein bands. (c) Elution profile of the SteD-SrcA complex examined by size exclusion chromatography-multiangle light scattering (SEC-MALS). The traces of MALS calculated molar mass (black, molar mass), light scattering (red, LS), UV absorbance (dotted green, UV) and differential refractive index (blue, RI) are shown. Calculated weight-averaged molar mass (Mw) is indicated below the black molar mass curve.