Dephosphorylation is the removal of a phosphate (PO43−) group by hydrolysis. To dephosphorylate a protein or DNA, an enzyme or hydrolase that cleaves ester bonds is required. For example, phosphatases remove phosphate groups by hydrolyzing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl (−OH) group.
Procedures for Dephosphorylation
Bovine Intestinal Alkaline Phosphatase (Product No. P4978)
10X CIP Buffer (Product No. C3225)
1 M NaCl
0.5 M Tris-HCl pH 7.9 at 25 °C
0.1 M MgCl2
0.01 M dithiothreitol
10 mM Tris-HCl, pH 8.2
50 mM KCl
1 mM MgCl2
0.1 mM ZnCl2
*Note: Phenol extraction or gel purification makes heat inactivation unnecessary.
Heat Inactivation: For the bovine intestinal enzyme, greater than 95% of the activity can be inactivated by heating to 75 °C for 10 minutes in the presence of 5 mM EDTA. Note: Alkaline phosphatase from E. coli is more heat stable than the bovine intestinal enzyme and is more resistant to heat inactivation.
Shrimp Alkaline Phosphatase (Product No. A2237) is supplied as a solution in 50% glycerol containing 25 mM Tris-HCl, pH 7.6, 1 mM MgCl2 and 0.1 mM ZnCl2.
Dilutions can be prepared in 0.05 M Tris-HCl, pH 8.5 containing 5 mM MgCl2.
Perform dephosphorylation in 0.05 M Tris-HCl, 5 mM MgCl2, pH 8.5.
Heat Inactivation: After the dephosphorylation reaction, the enzyme can then be inactivated by warming to 60 °C for 15 minutes.
Procedure: Incubate 100 units of alkaline phosphatase (Product No. P0114) with 400 µg of protein in 5 mM Tris pH 7.9, 10 mM NaCl, 1 mM MgCl2, and 0.1 mM DTT for 30 minutes at 30 °C.