Glucan phosphatases are essential for normal starch degradation in plants and glycogen metabolism in mammals. Arabidopsis thaliana phosphoglucan phosphatases starch excess 4 (SEX4) is one of fundamental repsentatives of a typical Dual Specificity Phosphatases (DSPs).

Carrillo et al. (DOI: 10.1016/j.ab.2016.11.005) report two methods on in-gel assays for glucan phosphatase detection. And SEX4 was selected as model which E .coli alkaline phosphatase (AP) was used as control. One method for detection of activity used pNPP and the second one applies BCIP/NBT. Firstly, the prepared gels were either stained with Coomassie Blue or developed for phosphatase activity using pNPP or the copule BCIP/NBT. Two phosphatases assayed showed a single, clear, easily visible chromogenic band which is coincident with the Coomassie Blue staining.

The sensitivity of the two methods was compared by increasing amount of the enzymes loaded to the native gels. For both methods, they found that band intensity is directly proportional to the mU of SEX4 enzyme loaded in each lane. The experimental results showed that the pNPP phosphatase assay is possible to detect up to 0.34 mU of SEX4 activity on the gels. And BCIP/NBT phosphatase assay is possible to detect up to 0.86 mU of SEX4 activity.

The work of Carrillo et al. demonstrated that pNPP and BCIP/NBT are very good substrates for visualization of glucan phosphatase activity enzymes after native PAGE gels since SEX4 enzyme was easily detected. And the comparison between pNPP and BCIP/NBT showed that pNPP would be a better substrate for detection of glucan phosphatases. And they speculate that two assays reported here could be extrapolated to test other glucan phosphatases.

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