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. 2003 Dec;185(24):7257–7265. doi: 10.1128/JB.185.24.7257-7265.2003

FIG. 2.

FIG. 2.

Purified BarA198 can autophosphorylate and transfer phosphate to SirA in vitro. (A) Purified SirA and BarA198 after nickel affinity chromatography, SDS-PAGE, and Coomassie blue staining. Both proteins are His6 tagged. Molecular mass markers (Bio-Rad prestained broad-range standards) are in the center lane (from bottom to top: 7.1, 21, 29, 35, 49, 80, 124, and 209 kDa). SirA-His6 is predicted to be 24.95 kDa; His6-BarA198 is predicted to be 81.60 kDa. (B) Time course of BarA198 autophosphorylation in the presence of [γ-32P]ATP. Reactions were allowed to proceed for the times indicated above each lane in minutes before aliquots were removed and the reactions were stopped by the addition of sample buffer containing SDS. Samples were resolved by SDS-PAGE and detected with a PhosphorImager. (C) Plot of band densities as determined by ImageQuant software from panel B. (D) Transphosphorylation of SirA by BarA198-P. BarA198 that had been preincubated with [γ-32P]ATP for 25 min was mixed with SirA as indicated. One reaction contained SirA with [γ-32P]ATP and no BarA198. Reactions were stopped at the times indicated above each lane in minutes by the addition of sample buffer containing SDS. Samples were resolved by SDS-PAGE and detected with a PhosphorImager. (E) Plot of band densities as determined by ImageQuant software from panel D. P, phosphorylated.