Wheat Germ Acid Phosphatase
Author David Williamson
Antarctic Phosphatase catalyzes the removal of 5´ phosphate groups from DNA and RNA. Since phosphatase-treated fragments lack the 5´ phosphoryl termini required by ligases, they cannot self-ligate. This property can be used to decrease the vector background in cloning strategies.
10 units of each phosphatase were incubated under recommended reaction conditions (including DNA) for 30 minutes and then heated at 65°C. Remaining phosphatase activity was measured by p-nitrophenyl-phosphate (pNPP) assay.
Phosphatases catalyze the hydrolysis of phosphate monoesters with consequent release of inorganic phosphate and the corresponding alcohol (or phenol in this case).
These enzymes are widely distributed in nature. Some essential points of enzyme studies are (1) to show the assay response is due to enzyme action, and (2) to show the response is dependent on conditions of the assay, such as pH and temperature.