Back to article: Direct detection of stringent alarmones (pp)pGpp using malachite green
FIGURE 2: Assessing the hydrolase activity of SpoT by using MG. (A) SpoT concentration dependent reduction of MG detection signal (A620nm). The SpoT reaction was given 90 minutes to occur before the MG detection mix was added. After 30 min incubation time, the absorbance (A620nm) was read via a plate reader. Control-2 contained the reaction components without ppGpp but with SpoT SYNoff protein. Control-1 is identical with the SpoT SYNoff samples except that SpoT SYNoff was added just before the reaction was stopped with MG. (B) A representative picture of the sample wells of (A). (C) (top) The scheme of the reactions that were performed. PPiase = pyrophosphatase. (bottom) Absorbance at 620 nm for the reactions as indicated above. (D, E, F) Hydrolysis of (p)ppGpp analogues, pGpp, ppGp, and pGp (each at 35.6 µM) by SpoT SYNoff (100 nM, 90 min) and their detection by MG. NC, the SpoT HYD reactions were similarly set up, but MG working reagent was added immediately to stop the HYD reaction. The mean ± SD of three technical replicates were plotted. Unpaired t-test was performed and the significance is displayed by the symbols * (p-value < 0.05), ** (p-value < 0.005).