Back to article: Direct detection of stringent alarmones (pp)pGpp using malachite green
FIGURE 1: Malachite green (MG) directly detects (p)ppGpp and its analogues. (A) Detection of potential SpoT HYD reaction products with MG. ppGpp was incubated (90 min, room temperature) with purified SpoT protein with only the HYD activity, i.e., SYNoff . Afterwards, MG reagent was added to the reaction mixes and read at 620 nm (A620nm). NC, sample: the two reactions with either heat inactivated or native SpoT SYNoff proteins, respectively. (B) Direct detection of the indicated chemicals by MG. The concentration of each compound was 35.6 µM (total volume 45 µl) before adding the MG detection mix. After 30 min incubation time, the absorbance at 620 nm (A620nm) was assessed. (C) Standard curve of detecting ppGpp via the MG mix. The data shown are the mean ± SD of three technical replicates except for (A), which is of two replicates. The significance of unpaired t-test is indicated by the symbols * (p-value < 0.05), *** (p-value < 0.001), and **** (p-value < 0.0001).