Dephosphorylation Procedures for DNA and Proteins

1. Dephosphorylation of DNA using Calf Intestinal Alkaline Phosphatase

Materials

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

Storage buffer

10 mM Tris-HCl, pH 8.2
50 mM KCl
1 mM MgCl₂
0.1 mM ZnCl₂
50% glycerol

Procedure:

1. Dissolve DNA in 1X CIP Buffer (0.5 µg DNA/10 µL).
2. For 5’ overhang DNA add 0.1 units/pmol CIP; for 3’ overhang or blunt end DNA add 1 unit/pmol.
3. Incubate 60 minutes at 37 °C.
4. Extract with phenol/chloroform² (Product No. P3803 or P2069) or gel purify the DNA.*
5. Recover the DNA by alcohol precipitation.²

*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.

2. Dephosphorylation of DNA Using Shrimp Alkaline Phosphatase

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.

3. Dephosphorylation of Protein using Bovine Intestinal Alkaline Phosphatase

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.