Dephosphorylation

Procedures for Dephosphorylation

Procedure	Recommended Product
1. Dephosphorylation of DNA using Calf Intestinal Alkaline Phosphatase	P4978
2. Dephosphorylation of DNA using Shrimp Alkaline Phosphatase	P9088
3. Dephosphorylation of Protein using Bovine Intestinal Alkaline Phosphatase	P0114

1. Dephosphorylation of DNA using Calf Intestinal Alkaline Phosphatase

Materials
Bovine Intestinal Alkaline Phosphatase, Cat. No. P4978
10X CIP Buffer (Cat. 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 (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.

1. Moessner,E. et al., Z. Physiol. Chem. 361, 543.
2. Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989), p 5.72, 6.22-6.47 and E.3-E.13.

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2. Dephosphorylation of DNA using Shrimp Alkaline Phosphatase

Shrimp Alkaline Phosphatase, Cat. No. P9088 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.

Dephosphorylation should be carried out 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.

1. Olsen, R., Overbo, K., Myrnes, B., Alkaline phosphatase from the hepatopancreas of shrimp (Pandalus borealis): a dimeric enzyme with catalytically active subunits, Comp. Biochem. Physiol., 99B, 755-761,1991.
2. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
3. Takanami, M., Analysis of the 5'-terminal nucleotide sequences of ribonucleic acids 1. the 5'-termini of Escherichia coli ribosomal RNA J. Mol. Biol. 23, 135 (1967).

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3. Dephosphorylation of Protein using Bovine Intestinal Alkaline Phosphatase

Procedure:

1. Incubate 100 units of alkaline phosphatase, Cat. No. P0114 with 400 ug 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.
2. For SDS PAGE analysis, the reaction can be stopped with the addition of 5X sample buffer and boiling.

1. Labugger R, et al. Extensive Troponin I and T Modification Detected in Serum from Patients with Acute Myocardial Infarction. Circulation 102(11): 1221-1226 (2000).
   

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