In each sample, reaction is stopped by adding 20 μl of malachite green reagent for subsequent determination of the quantity of phosphate released. To this aim, absorbance in each sample is read at 620 nm in a Safire2 UV spectrophotometer microplate reader after 20 min of incubation at room temperature (Figure 2B). Values are then compared with a standard curve of free phosphate prepared according to the malachite green phosphate assay kit manufacturer’s instructions (Figure 3).
Figure 3. Use of the malachite green phosphate assay kit to determine free phosphate concentrations. A. Color formation was observed after 20 min of incubation at room temperature for different concentrations of phosphate (from well 1 to 8, 2 wells for each concentration): 0 μM, 4 μM, 8 μM, 12 μM, 16 μM, 24 μM, 32 μM and 40 μM (buffer only, 100 mM Tris-HCl pH 7.5, 5 mM MgCl
2, final concentrations). B. Standard curve representing absorbance monitored at 620 nm as a function of variable quantities of free phosphate (Pi). In this example, the linear regression curve was Abs 620 nm = [0.0001 x (pmol Pi in 100 μl)] -0.0085, and R
2 = 0.9985. C. Table corresponding to values of absorbance measured for different quantities of free phosphate.